Diagnostic and prognostic methods for lung disorders using gene expression profiles from nose epithelial cells

ABSTRACT

The present invention provides methods for diagnosis and prognosis of lung cancer using expression analysis of one or more groups of genes, and a combination of expression analysis from a nasal epithelial cell sample. The methods of the invention provide far less invasive method with a superior detection accuracy for lung cancer when compared to any other currently available method for lung cancer diagnostic or prognosis. The invention also provides methods of diagnosis and prognosis of other lung diseases, such as lung cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. 119(e) fromU.S. provisional application Ser. No. 60/780,552, filed on Mar. 9, 2006,the content of which is herein incorporated by reference in itsentirety.

GOVERNMENT SUPPORT

The present invention was made, in part, by support from the NationalInstitutes of Health grant No. HL077498. The United States Governmenthas certain rights to the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to methods for diagnosing lungdiseases from nasal epithelial cells using gene expression analysis.More specifically, the invention is directed to diagnostic andprognostic methods for detecting from nasal epithelial cell samples lungdiseases, particularly lung cancer in subjects, preferably humans. Theinvention also provides genes the expression of which can be used toanalyze lung diseases from the nasal epithelial cell samples.

2. Background

Lung disorders represent a serious health problem in the modern society.For example, lung cancer claims more than 150,000 lives every year inthe United States, exceeding the combined mortality from breast,prostate and colorectal cancers. Cigarette smoking is the mostpredominant cause of lung cancer. Presently, 25% of the U.S. populationsmokes, but only 10% to 15% of heavy smokers develop lung cancer. Thereare also other disorders associated with smoking such as emphysema.There are also health questions arising from people exposed to smokers,for example, second hand smoke. Former smokers remain at risk fordeveloping such disorders including cancer and now constitute a largereservoir of new lung cancer cases. In addition to cigarette smoke,exposure to other air pollutants such as asbestos, and smog, pose aserious lung disease risk to individuals who have been exposed to suchpollutants.

Approximately 85% of all subjects with lung cancer die within threeyears of diagnosis. Unfortunately survival rates have not changedsubstantially over the past several decades. This is largely becausethere are no effective methods for identifying smokers who are athighest risk for developing lung cancer and no effective tools for earlydiagnosis.

The methods that are currently employed to diagnose lung cancer includechest X-ray analysis, bronchoscopy or sputum cytological analysis,computer tomographic analysis of the chest, and positron electrontomographic (PET) analysis. However, none of these methods provide acombination of both sensitivity and specificity needed for an optimaldiagnostic test.

We have previously found that a gene group expression pattern analysisfrom biological samples taken from bronchial epithelial cells permitsaccurate method for diagnosis and prognosis for development of lungdiseases, such as lung cancer (PCT/US2006/014132).

However, the method of sampling epithelial cells from bronchial tissuewhile less invasive than many other methods has some drawbacks. Forexample, the patient may not eat or drink for about 6-12 hours prior tothe test. Also, if the procedure is performed using a rigid bronchoscopethe patient needs general anesthesia involving related risks to thepatient. When the method is performed using a flexible bronchoscope, theprocedure is performed using local anesthesia. However, several patientsexperience uncomfortable sensations, such as a sensation of suffocatingduring such a procedure and thus are relatively resistant for goingthrough the procedure more than once. Also, after the bronchoscopyprocedure, the throat may feel uncomfortably scratchy for several days.

While it has been previously described, that RNA can be isolated frommouth epithelial cells for gene expression analysis (U.S. Ser. No.10/579,376), it has not been clear if such samples routinely reflect thesame gene expression changes as bronchial samples that can be used inaccurate diagnostic and prognostic methods.

Thus, there is significant interest and need in developing simplenon-invasive screening methods for assessing an individual's lungdisease, such as lung cancer or risk for developing lung cancer,including primary lung malignancies. It would be preferable if such amethod would be more accurate than the traditional chest x-ray or PETanalysis or cytological analysis, for example by identifying markergenes which have their expression altered at various states of diseaseprogression.

Therefore, the development of non-invasive tests would be very helpful.

SUMMARY OF THE INVENTION

The present invention provides a much less invasive method fordiagnosing lung diseases, such as lung cancer based on analysis of geneexpression in nose epithelial cells.

We have found surprisingly that the gene expression changes in noseepithelial cells closely mirrors the gene expression changes in the lungepithelial cells. Accordingly, the invention provides methods fordiagnosis, prognosis and follow up of progression or success oftreatment for lung diseases using gene expression analysis from noseepithelial cells.

We have also found that the gene expression pattern in the bronchialepithelial cells and nasal epithelial cells very closely correlated.This is in contrast with epithelial cell expression pattern in any othertissue we have studies thus far. The genes the expression of which isparticularly closely correlated between the lung and the nose are listedin tables 8, 9 and 10.

The method provides an optimal means for screening for changesindicating malignancies in individuals who, for example are at risk ofdeveloping lung diseases, particularly lung cancers because they havebeen exposed to pollutants, such as cigarette or cigar smoke or asbestosor any other known pollutant. The method allows screening at a routineannual medical examination because it does not need to be performed byan expert trained in bronchoscopy and it does not require sophisticatedequipment needed for bronchoscopy.

We discovered that there is a significant correlation between theepithelial cell gene expression in the brinchial tissue and in the nasalpassages. We discovered this by analyzing samples from individuals withcancer as well as by analyzing samples from smokers compared tonon-smokers.

We discovered a strong correlation between the gene expression profilein the bronchial and nasal epithelial cell samples when we analyzedgenes that distinguish individuals with known sarcoidosis fromindividuals who do not have sarcoidosis.

We also discovered that the same is true, when one compares the changesin the gene expression pattern between smokers and individuals who havenever smoked.

Accordingly, we have found a much less invasive method of sampling forprognostic, diagnostic and follow-up purposes by taking epithelialsamples from the nasal passages as opposed to bronchial tissue, and thatthe same genes that have proven effective predictors for lung diseases,such as lung cancer, in smokers and non-smokers, can be used in analysisof epithelial cells from the nasal passages.

The gene expression analysis can be performed using genes and/or groupsof genes as described in tables 8, 9 and 10 and, for example, inPCT/US2006/014132. Naturally, other diagnostic genes may also be used,as they are identified.

Accordingly, the invention provides a substantially less invasive methodfor diagnosis, prognosis, and follow-up of lung diseases using samplesfrom nasal epithelial cells. To provide an improved analysis, onepreferably uses gene expression analysis.

One can use analysis of gene transcripts individually and in groups orsubsets for enhanced diagnosis for lung diseases, such as lung cancer.

Similarly, as the art continues to identify the gene expression changesassociated with other lung diseases wherein the disease causes a fieldeffect, namely, wherein the disease-causing agent, i.e. a pollutant, ora microbe or other airway irritant, the analysis and discoveriespresented herein allow us to conclude that those gene expression changescan also be analyzed from nasal epithelial cells thus providing a muchless invasive and more accurate method for diagnosing lung diseases ingeneral. For example, using the methods as described, one can diagnoseany lung disease that results in detectable gene expression changes,including, but not limited to acute pulmonary eosinophilia (Loeffler'ssyndrome), CMV pneumonia, chronic pulmonary coccidioidomycosis,cryptococcosis, disseminated tuberculosis (infectious), chronicpulmonary histoplasmosis, pulmonary actinomycosis, pulmonaryaspergilloma (mycetoma), pulmonary aspergillosis (invasive type),pulmonary histiocytosis X (eosinophilic granuloma), pulmonarynocardiosis, pulmonary tuberculosis, and sarcoidosis. In fact, one ofthe examples shows a group of genes the expression of which changes whenthe individual is affected with sarcoidosis.

One example of the gene transcript groups useful in thediagnostic/prognostic tests of the invention using nasal epithelialcells are set forth in Table 6. We have found that taking groups of atleast 20 of the Table 6 genes provides a much greater diagnosticcapability than chance alone.

Preferably one would use more than 20 of these gene transcript, forexample about 20-100 and any combination between, for example, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, and so on. Our preferred groups are thegroups of 361 (Table 8), 107 (Table 9), 70 (Table 10), 96 (Table 1), 84(Table 2), 50 (Table 3), 36 (Table 4), 80 (Table 5), 535 (Table 6) and20 (Table 7).

In some instances, we have found that one can enhance the accuracy ofthe diagnosis by adding certain additional genes to any of thesespecific groups. When one uses these groups, the genes in the group arecompared to a control or a control group. The control groups can beindividuals who have not been exposed to a particular airway irritant,such as non-smokers, smokers, or former smokers, or individuals notexposed to viruses or other substance that can cause a “filed effect” inthe airways thus resulting in potential for lung disease. Typically,when one wishes to diagnose a disease, the control sample should be froman individual who does not have the diseases and alternatively includeone or more samples with individuals who have similar or different lungdiseases. Thus, one can match the sample one wishes to diagnose with acontrol wherein the expression pattern most closely resembles theexpression pattern in the sample. Preferably, one compares the genetranscripts or their expression product in the biological sample of anindividual against a similar group, except that the members of thecontrol groups do not have the lung disorder, such as emphysema or lungcancer. For example, comparing can be performed in the biological samplefrom a smoker against a control group of smokers who do not have lungcancer. When one compares the transcripts or expression products againstthe control for increased expression or decreased expression, whichdepends upon the particular gene and is set forth in the tables—not allthe genes surveyed will show an increase or decrease. However, at least50% of the genes surveyed must provide the described pattern. Greaterreliability is obtained as the percent approaches 100%. Thus, in oneembodiment, one wants at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 98%, or 99% of the genes surveyed to show the altered patternindicative of lung disease, such as lung cancer, as set forth in thetables, infra.

In one embodiment, the nasal epithelial cell sample is analyzed for agroup of genes the expression of which is altered in individuals who areat risk of developing lung diseases, such as lung cancer, because of theexposure to air pollutants or other airway irritant such as microbesthat occur in the air and are inhaled. This is because we havediscovered that air pollutant The method can also be used for analysisof groups of genes the expression of which is consistently altered as agroup in individuals who are at risk of developing lung diseases becauseof the exposure to such air pollutants including microbes and virusespresent in the air.

One can analyze the nasal epithelial cells according to the methods ofthe present invention using gene groups the expression pattern orprofile of which can be used to diagnose lung diseases, such as lungcancer and even the type of lung cancer, in more than 60%, preferablymore than 65%, still more preferably at least about 70%, still morepreferably about 75%, or still more preferably about 80%-95% accuracyfrom a sample taken from airways of an individual screened for a lungdisease, such as lung cancer.

In one embodiment, the invention provides a method of diagnosing a lungdisease such as lung cancer using a combination of nasal epithelialcells and the analysis of gene expression pattern of the gene groups asdescribed in the present invention.

Accordingly, the invention provides methods for analyzing gene groupsfrom nasal epithelial cells, wherein the gene expression pattern thatcan be directly used in diagnosis and prognosis of lung diseases.Particularly, the invention provides analysis from nasal epithelialcells groups of genes the expression profile of which provides adiagnostic and or prognostic test to determine lung disease in anindividual exposed to air pollutants. For example, the inventionprovides analysis from nasal epithelial cells, groups of genes theexpression profile of which can distinguish individuals with lung cancerfrom individuals without lung cancer.

In one embodiment, the invention provides an early asymptomaticscreening system for lung cancer by using the analysis of nasalepithelial cells for the disclosed gene expression profiles. Suchscreening can be performed, for example, in similar age groups ascolonoscopy for screening colon cancer. Because early detection in lungcancer is crucial for efficient treatment, the gene expression analysissystem of the present invention provides an improved method to detecttumor cells. Thus, the analysis can be made at various time intervals,such as once a year, once every other year for screening purposes.Alternatively, one can use a more frequent sampling if one wishes tomonitor disease progression or regression in response to a therapeuticintervention. For example, one can take samples from the same patientonce a week, once or two times a month, every 3, 4, 5, or 6 months.

The probes that can be used to measure expression of the gene groups ofthe invention can be nucleic acid probes capable of hybridizing to theindividual gene/transcript sequences identified in the presentinvention, or antibodies targeting the proteins encoded by theindividual gene group gene products of the invention. The probes arepreferably immobilized on a surface, such as a gene or protein chip soas to allow diagnosis and prognosis of lung diseases in an individual.

In one preferred embodiment, the invention provides a group of genesthat can be used in diagnosis of lung diseases from the nasal epithelialcells. These genes were identified using

In one embodiment, the invention provides a group of genes that can beused as individual predictors of lung disease. These genes wereidentified using probabilities with a t-test analysis and showdifferential expression in smokers as opposed to non-smokers. The groupof genes comprise ranging from 1 to 96, and all combinations in between,for example 5, 10, 15, 20, 25, 30, for example at least 36, at leastabout, 40, 45, 50, 60, 70, 80, 90, or 96 gene transcripts, selected fromthe group consisting of genes identified by the following GenBanksequence identification numbers (the identification numbers for eachgene are separated by “;” while the alternative GenBank ID numbers areseparated by “///”): NM_(—)003335; NM_(—)000918; NM_(—)006430.1;NM_(—)001416.1; NM_(—)004090; NM_(—)006406.1; NM_(—)003001.2;NM_(—)001319; NM_(—)006545.1; NM_(—)021145.1; NM_(—)002437.1;NM_(—)006286; NM_(—)001003698///NM_(—)001003699///NM_(—)002955;NM_(—)001123///NM_(—)006721; NM_(—)024824; NM_(—)004935.1;NM_(—)002853.1; NM_(—)019067.1; NM_(—)024917.1; NM_(—)020979.1;NM_(—)005597.1; NM_(—)007031.1; NM_(—)009590.1; NM_(—)020217.1;NM_(—)025026.1; NM_(—)014709.1; NM_(—)014896.1; AF010144;NM_(—)005374.1; NM_(—)001696; NM_(—)005494///NM_(—)058246;NM_(—)006534///NM_(—)181659; NM_(—)006368; NM_(—)002268///NM_(—)032771;NM_(—)014033; NM_(—)016138; NM_(—)007048///NM_(—)194441; NM_(—)006694;NM_(—)000051///NM_(—)138292///NM_(—)138293;NM_(—)000410///NM_(—)139002///NM_(—)139003///NM_(—)139004///NM_(—)139005///NM_(—)139006///NM_(—)139007///NM_(—)139008///NM_(—)139009///NM_(—)139010///NM_(—)139011;NM_(—)004691; NM_(—)012070///NM_(—)139321///NM_(—)139322; NM_(—)006095;AI632181; AW024467; NM_(—)021814; NM_(—)005547.1; NM_(—)203458;NM_(—)015547///NM_(—)147161; AB007958.1; NM_(—)207488;NM_(—)005809///NM_(—)181737///NM_(—)181738; NM_(—)016248///NM_(—)144490;AK022213.1; NM_(—)005708; NM 207102; AK023895;NM_(—)144606///NM_(—)144997; NM_(—)618530; AK021474; U43604.1; AU147017;AF222691.1; NM_(—)015116;NM_(—)001005375///NM_(—)001005785///NM_(—)001005786///NM_(—)004081///NM_(—)020363///NM_(—)020364///NM_(—)020420;AC004692; NM_(—)001014; NM_(—)000585///NM_(—)172174///NM_(—)172175;NM_(—)054020///NM_(—)172095///NM_(—)172096///NM_(—)172097; BE466926;NM_(—)018011; NM_(—)024077; NM_(—)012394;NM_(—)019011///NM_(—)207111///NM_(—)207116; NM_(—)017646; NM_(—)021800;NM_(—)016049; NM_(—)014395; NM_(—)014336; NM_(—)018097; NM_(—)019014;NM_(—)024804; NM_(—)018260; NM_(—)018118; NM_(—)014128; NM_(—)024084;NM_(—)005294; AF077053; NM_(—)138387; NM_(—)024531; NM_(—)000693;NM_(—)018509; NM_(—)033128; NM_(—)020706; AI523613; and NM_(—)014884,the expression profile of which can be used to diagnose lung disease,for example lung cancer, in lung cell sample from a smoker, when theexpression pattern is compared to the expression pattern of the samegroup of genes in a smoker who does not have or is not at risk ofdeveloping lung cancer.

In another embodiment, the gene/transcript analysis comprises a group ofabout 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80, 80-90,90-100, 100-120, 120-140, 140-150, 150-160, 160-170, 170-180, 180-190,190-200, 200-210, 210-220, 220-230, 230-240, 240-250, 250-260, 260-270,270-280, 280-290, 290-300, 300-310, 310-320, 320-330, 330-340, 340-350,350-360, 360-370, 370-380, 380-390, 390-400, 400-410, 410-420, 420-430,430-440, 440-450, 450-460, 460-470, 470-480, 480-490, 490-500, 500-510,510-520, 520-530, and up to about 535 genes selected from the groupconsisting of genes or transcripts as shown in the Table 6.

In one embodiment, the genes are selected from the group consisting ofgenes or transcripts as shown in Table 5.

In another embodiment, the genes are selected from the genes ortranscripts as shown in Table 7.

In one embodiment, the transcript analysis gene group comprises a groupof individual genes the change of expression of which is predictive of alung disease either alone or as a group, the gene transcripts selectedfrom the group consisting of NM_(—)007062.1; NM_(—)001281.1; BC002642.1;NM_(—)000346.1; NM_(—)006545.1; BG034328; NM_(—)019067.1;NM_(—)017925.1; NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1;NM_(—)002268///NM_(—)032771; NM_(—)007048///NM_(—)194441; NM_(—)006694;U85430.1; NM_(—)004691; AB014576.1; BF218804; BE467941; R83000;AL161952.1; AK023843.1; AK021571.1; AK023783.1; AL080112.1; AW971983;AI683552; NM_(—)024006.1; AK026565.1; NM_(—)014182.1; NM_(—)021800.1;NM_(—)016049.1; NM_(—)021971.1; NM_(—)014128.1; AA133341; AF198444.1.

In one embodiment, the gene group comprises a probe set capable ofspecifically hybridizing to at least all of the 36 gene products. Geneproduct can be mRNA which can be recognized by an oligonucleotide ormodified oligonucleotide probe, or protein, in which case the probe canbe, for example an antibody specific to that protein or an antigenicepitope of the protein.

In yet another embodiment, the invention provides a gene group, whereinthe expression pattern of the group of genes provides diagnostic for alung disease. The gene group comprises gene transcripts encoded by agene group consisting of at least for example 5, 10, 15, 20, 25, 30,preferably at least 36, still more preferably 40, still more preferably45, and still more preferably 46, 47, 48, 49, or all 50 of the genesselected from the group consisting of and identified by their GenBankidentification numbers: NM_(—)007062.1; NM_(—)001281.1; BC000120.1;NM_(—)014255.1; BC002642.1; NM_(—)000346.1; NM_(—)006545.1; BG034328;NM_(—)021822.1; NM_(—)021069.1; NM_(—)019067.1; NM_(—)017925.1;NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1; AF126181.1; U 93240.1;U90552.1; AF151056.1; U85430.1; U51007.1; BC005969.1; NM_(—)002271.1;AL566172; AB014576.1; BF218804; AK022494.1; AA114843; BE467941;NM_(—)003541.1; R83000; AL161952.1; AK023843.1; AK021571.1; AK023783.1;AU147182; AL080112.1; AW971983; AI683552; NM_(—)024006.1; AK026565.1;NM_(—)014182.1; NM_(—)021800.1; NM_(—)016049.1; NM_(—)019023.1;NM_(—)021971.1; NM_(—)014128.1; AK025651.1; AA133341; and AF198444.1. Inone preferred embodiment, one can use at least 20 of the 36 genes thatoverlap with the individual predictors and, for example, 5-9 of thenon-overlapping genes and combinations thereof.

In another embodiment, the invention provides a group of about 30-180,preferably, a group of about 36-150 genes, still more preferably a groupof about 36-100, and still more preferably a group of about 36-50 genes,the expression profile of which is diagnostic of lung cancer inindividuals who smoke.

In one embodiment, the invention provides a group of genes theexpression of which is decreased in an individual having lung cancer. Inone embodiment, the group of genes comprises at least 5-10, 10-15,15-20, 20-25 genes selected from the group consisting of NM_(—)000918;NM_(—)006430.1; NM_(—)001416.1; NM_(—)004090; NM_(—)006406.1;NM_(—)003001.2; NM_(—)006545.1; NM_(—)002437.1-NM_(—)006286;NM_(—)001123///NM_(—)006721; NM_(—)024824; NM_(—)004935.1; NM_(—)001696;NM_(—)005494///NM_(—)058246; NM_(—)006368; NM_(—)002268///NM_(—)032771;NM_(—)006694; NM_(—)004691; NM_(—)012394; NM_(—)021800; NM_(—)016049;NM_(—)138387; NM_(—)024531; and NM_(—)018509. One or more other genescan be added to the analysis mixtures in addition to these genes.

In another embodiment, the group of genes comprises genes selected fromthe group consisting of NM_(—)014182.1; NM_(—)001281.1; NM_(—)024006.1;AF135421.1; L76200.1; NM_(—)000346.1; BC008710.1; BC000423.2;BC008710.1; NM_(—)007062; BC075839.1///BC073760.1;BC072436.1///BC004560.2; BC001016.2; BC005023.1; BC000360.2; BC007455.2;BC023528.2///BC047680.1; BC064957.1; BC008710.1; BC066329.1; BC023976.2;BC008591.2///BC050440.1///BC048096.1; and BC028912.1.

In yet another embodiment, the group of genes comprises genes selectedfrom the group consisting of NM_(—)007062.1; NM_(—)001281.1; BC000120.1;NM_(—)014255.1; BC002642.1; NM_(—)000346.1, NM_(—)006545.1; BG034328;NM_(—)021822.1; NM_(—)021069.1; NM_(—)019067.1; NM_(—)017925.1;NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1; AF126181.1; U93240.1;U90552.1; AF151056.1; U85430.1; U51007.1; BC005969.1; NM_(—)002271.1;AL566172; and AB014576.1.

In one embodiment, the invention provides a group of genes theexpression of which is increased in an individual having lung cancer. Inone embodiment, the group of genes comprises genes selected from thegroup consisting of NM_(—)003335; NM_(—)001319; NM_(—)021145.1;NM_(—)001003698///NM_(—)001003699///; NM_(—)002955; NM_(—)002853.1;NM_(—)019067.1; NM_(—)024917.1; NM_(—)020979.1; NM_(—)005597.1;NM_(—)007031.1; NM_(—)009590.1; NM_(—)020217.1; NM_(—)025026.1;NM_(—)014709.1; NM_(—)014896.1; AF010144; NM_(—)005374.1;NM_(—)006534///NM_(—)81659; NM_(—)014033; NM_(—)016138;NM_(—)007048///NM_(—)194441; NM_(—)000051///NM_(—)138292///NM_(—)138293;NM_(—)000410///NM_(—)139002///NM_(—)139003///NM_(—)139004///NM_(—)139005///NM_(—)139006///NM_(—)139007///NM_(—)139008///NM_(—)139009///NM_(—)139010///NM_(—)139011;NM_(—)012070///NM_(—)139321///NM_(—)139322; NM_(—)006095; AI632181;AW024467; NM_(—)021814; NM_(—)005547.1; NM_(—)203458;NM_(—)015547///NM_(—)147161; AB007958.1; NM_(—)207488;NM_(—)005809///NM_(—)181737///NM_(—)181738; NM_(—)016248///NM_(—)144490;AK022213.1; NM_(—)005708; NM_(—)207102; AK023895;NM_(—)144606///NM_(—)144997; NM_(—)018530; AK021474; U43604.1; AU147017;AF222691.1; NM_(—)015116;NM_(—)001005375///NM_(—)001005785///NM_(—)001005786///NM_(—)004081///NM_(—)020363///NM_(—)020364///NM_(—)020420;AC004692; NM_(—)001014; NM_(—)000585///NM_(—)172174///NM_(—)172175;NM_(—)054020///NM_(—)172095///NM_(—)172096///NM_(—)172097; BE466926;NM_(—)018011; NM_(—)024077; NM_(—)019011///NM_(—)20711///NM_(—)207116;NM_(—)017646; NM_(—)014395; NM_(—)014336; NM_(—)018097; NM_(—)019014;NM_(—)024804; NM_(—)018260; NM_(—)018118; NM_(—)014128; NM_(—)024084;NM_(—)005294; AF077053; NM_(—)000693; NM_(—)033128; NM_(—)020706;AI523613; and NM_(—)014884.

In one embodiment, the group of genes comprises genes selected from thegroup consisting of NM_(—)030757.1; R83000; AK021571.1; NM_(—)17932.1;U85430.1; AI683552; BC002642.1; AW024467; NM_(—)030972.1; BC021135.1;AL161952.1; AK026565.1; AK023783.1; BF218804; AK023843.1; BC001602.1;BC034707.1; BC064619.1; AY280502.1; BC059387.1; BC061522.1; U50532.1;BC006547.2; BC008797.2; BC000807.1; AL080112.1;BC033718.1///BC046176.1///; BC038443.1; Hs.288575 (UNIGENE ID);AF020591.1; BC002503.2; BC009185.2; Hs.528304 (UNIGENE ID); U50532.1;BC013923.2; BC031091; Hs.249591 (Unigene ID); Hs.286261 (Unigene ID);AF348514.1; BC066337.1///BC058736.1///BC050555.1; Hs.216623 (UnigeneID); BC072400.1; BC041073.1; U43965.1; BC021258.2; BC016057.1;BC016713.11//BC014535.1///AF237771.1; BC000701.2; BC010067.2; Hs.156701(Unigene ID); BC030619.2; U43965.1; Hs.438867 (Unigene ID);BC035025.2///BC050330.1; BC074852.2///BC074851.2; Hs.445885 (UnigeneID); AF365931.1; and AF257099.1.

In one embodiment, the group of genes comprises genes selected from thegroup consisting of BF218804; AK022494.1; AA114843; BE467941;NM_(—)003541.1; R83000; AL161952.1; AK023843.1; AK021571.1; AK023783.1;AU147182; AL080112.1; AW971983; AI1683552; NM_(—)024006.1; AK026565.1;NM_(—)014182.1; NM_(—)021800.1; NM_(—)016049.1; NM_(—)019023.1;NM_(—)021971.1; NM_(—)014128.1; AK025651.1; AA133341; and AF198444.1.

In another embodiment, the invention provides a method for diagnosing alung disease comprising obtaining a nucleic acid sample from lung,airways or mouth of an individual exposed to an air pollutant, analyzingthe gene transcript levels of one or more gene groups provided by thepresent invention in the sample, and comparing the expression pattern ofthe gene group in the sample to an expression pattern of the same genegroup in an individual, who is exposed to similar air pollutant but nothaving lung disease, such as lung cancer or emphysema, wherein thedifference in the expression pattern is indicative of the testindividual having or being at high risk of developing a lung disease.The decreased expression of one or more of the genes, preferably all ofthe genes including the genes listed on Tables 1-4 as “down” whencompared to a control, and/or increased expression of one or more genes,preferably all of the genes listed on Tables 1-4 as “up” when comparedto an individual exposed to similar air pollutants who does not have alung disease, is indicative of the person having a lung disease or beingat high risk of developing a lung disease, preferably lung cancer, inthe near future and needing frequent follow ups to allow early treatmentof the disease.

In one preferred embodiment, the lung disease is lung cancer. In oneembodiment, the air pollutant is tobacco or tobacco smoke.

Alternatively, the diagnosis can separate the individuals, such assmokers, who are at lesser risk of developing lung diseases, such aslung cancer by analyzing from the nasal epithelial cells the expressionpattern of the gene groups of the invention provides a method ofexcluding individuals from invasive and frequent follow ups.

Accordingly, in one embodiment, the invention provides methods forprognosis, diagnosis and therapy designs for lung diseases comprisingobtaining an nasal epithelial cell sample from an individual who smokesand analyzing expression profile of the gene groups of the presentinvention, wherein an expression pattern of the gene group that deviatesfrom that in a healthy age, race, and gender matched smoker, isindicative of an increased risk of developing a lung disease. Tables 1-4indicate the expression pattern differences as either being down or upas compared to a control, which is an individual exposed to similarairway pollutant but not affected with a lung disease.

The invention also provides methods for prognosis, diagnosis and therapydesigns for lung diseases comprising obtaining an nasal epithelial cellsample from a non-smoker individual and analyzing expression profile ofthe gene groups of the present invention, wherein an expression patternof the gene group that deviates from that in a healthy age, race, andgender matched smoker, is indicative of an increased risk of developinga lung disease.

In one embodiment, the analysis is performed using nucleic acids,preferably RNA, in the biological sample.

In one embodiment, the analysis is performed analyzing the amount ofproteins encoded by the genes of the gene groups of the inventionpresent in the sample.

In one embodiment the analysis is performed using DNA by analyzing thegene expression regulatory regions of the groups of genes of the presentinvention using nucleic acid polymorphisms, such as single nucleic acidpolymorphisms or SNPs, wherein polymorphisms known to be associated withincreased or decreased expression are used to indicate increased ordecreased gene expression in the individual. For example, methylationpatterns of the regulatory regions of these genes can be analyzed.

In one embodiment, the present invention provides a minimally invasivesample procurement method for obtaining nasal epithelial cell RNA thatcan be analyzed by expression profiling of the groups of genes, forexample, by array-based gene expression profiling. These methods can beused to diagnose individuals who are already affected with a lungdisease, such as lung cancer, or who are at high risk of developing lungdisease, such as lung cancer, as a consequence of being exposed to airpollutants. These methods can also be used to identify further patternsof gene expression that are diagnostic of lung disorders/diseases, forexample, cancer or emphysema, and to identify subjects at risk fordeveloping lung disorders.

The invention further provides a method of analyzing nasal epithelialcells using gene group microarray consisting of one or more of the genegroups provided by the invention, specifically intended for thediagnosis or prediction of lung disorders or determining susceptibilityof an individual to lung disorders.

In one embodiment, the invention relates to a method of diagnosing adisease or disorder of the lung comprising obtaining a sample from nasalepithelial cells, wherein the sample is a nucleic acid or proteinsample, from an individual to be diagnosed; and determining theexpression of group of identified genes in said sample, wherein changedexpression of such gene compared to the expression pattern of the samegene in a healthy individual with similar life style and environment isindicative of the individual having a disease of the lung.

In one embodiment, the invention relates to a method of diagnosing adisease or disorder of the lung comprising obtaining at least two nasalepithelial samples, wherein the samples are either nucleic acid orprotein samples, in at least one, two, 3, 4, 5, 6, 7, 8, 9, or more timeintervals from an individual to be diagnosed; and determining theexpression of the group of identified genes in said sample, whereinchanged expression of at least about for example 5, 10, 15, 20, 25, 30,preferably at least about 36, 40, 50, 60, 70, 80, 90, 100, 110, 120,130, 140, 150, 160, 170, or 180 of such genes in the sample taken laterin time compared to the sample taken earlier in time is diagnostic of alung disease.

In one embodiment, the disease of the lung is selected from the groupconsisting of asthma, chronic bronchitis, emphysema, primary pulmonaryhypertension, acute respiratory distress syndrome, hypersensitivitypneumonitis, eosinophilic pneumonia, persistent fungal infection,pulmonary fibrosis, systemic sclerosis, idiopathic pulmonaryhemosiderosis, pulmonary alveolar proteinosis, and lung cancer, such asadenocarcinoma, squamous cell carcinoma, small cell carcinoma, largecell carcinoma, and benign neoplasm of the lung (e.g., bronchialadenomas and hamartomas).

In a particular embodiment, the nucleic acid sample is RNA.

In one embodiment, individual to be diagnosed is an individual who hasbeen exposed to tobacco smoke, an individual who has smoked, or anindividual who currently smokes.

The invention also provides analysis of nasal epithelial cells using anarray, for example, a microarray for diagnosis of a disease of the lunghaving immobilized thereon a plurality of oligonucleotides whichhybridize specifically to genes of the gene groups which aredifferentially expressed in airways exposed to air pollutants, such ascigarette smoke, and have or are at high risk of developing lungdisease, as compared to those individuals who are exposed to similar airpollutants and airways which are not exposed to such pollutants. In oneembodiment, the oligonucleotides hybridize specifically to one allelicform of one or more genes which are differentially expressed for adisease of the lung. In a particular embodiment, the differentiallyexpressed genes are selected from the group consisting of the genesshown in tables 1-4; preferably the group of genes comprises genesselected from the Table 3. In one preferred embodiment, the group ofgenes comprises the group of at least 20 genes selected from Table 3 andadditional 5-10 genes selected from Tables 1 and 2. In one preferredembodiment, at least about 10 genes are selected from Table 4.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows hierarchical clustering of bronchial airway epithelialsamples from current (striped box) and never (white box) smokersaccording to the expression of 60 genes whose expression levels arealtered by smoking in the nasal epithelium. Airway samples tend to groupwith their appropriate class. Dark grey indicates higher level ofexpression and light grey lower level of expression.

FIG. 2 shows hierarchical clustering of nasal epithelial samples frompatients with sarcoid (stiped box) and normal healthy volunteers (whitebox) according to the expression of top 20 t-test genes that differbetween the 2 groups (P<0.00005). With few exceptions, samples groupinto their appropriate classes. Light grey=low level of expression,black=mean level of expression, dark grey=high level of expression.

FIG. 3 shows smoking related genes in mouth, nose and bronchus.Principal component analysis (PCA) shows the variation in expression ofgenes affected by tobacco exposure in current smokers (dark grey) andnever smokers (black). Airway epithelium type is indicated by the symbolshape: bronchial (circle), nasal (triangle) and mouth (square). Sampleslargely separate by smoking status across the first principal component,with the exception of samples from mouth. This indicates a common geneexpression host response that can be seen both in the bronchialepithelial tissue and the nasal epithelial tissue.

FIG. 4 shows a supervised hierarchical clustering analysis of cancersamples. Individuals with sarcoidosis and individuals with no sarcoidswere sampled from both lung tissues and nasal tissues. Gene expressionanalysis showed that expression of 37 genes can be used to differentiatethe cancer samples and non-cancer sampled either from bronchial or nasalepithelial cells. Light grey in the clustering analysis indicates lowlevel of expression and dark grey high level of expression. Asterisknext to the circles indicates that these samples were from an individualwith stage 0-1 sarcoidosis. The dot next to the circle indicates thatthese samples were from an individual with a stage 4 sarcoidosis.

FIG. 5 shows airway t-test genes projected on nose data including the107 leading edge genes as shown in Table 9. Enrichment of differentiallyexpressed bronchial epithelial genes among genes highly changed in thenasal epithelium in response to smoking. Results from GSEA analysisshows the leading edge of the set of 361 differentially expressedbronchial epithelial genes being overrepresented among the top rankedlist of genes differentially expressed in nasal epithelium cells inresponse to smoking. There are 107 genes that comprise the “leading edgesubset” (p<0.001).

FIG. 6 shows 107 Leading Edge Genes from Airway—PCA on Nose Samples.Asterisk next to the circle indicates current smokers. Dark circlesrepresent samples from never smokers. Principal component analysis of107 “leading edge” genes from bronchial epithelial cells enriched in thenasal epithelial gene expression profile. Two dimensional PCA of the 107“leading edge” genes from the bronchial epithelial signature that areenriched in the nasal epithelial cell expression profile.

FIG. 7 shows a Bronch projection from 10 tissues. From this figure onecan see, that the samples from bronchial epithelial cells (dottedsquares) and the samples from nose epithelial cells (crossed squares)overlapped closely and were clearly distinct from samples from othertissues, including mouth. Principal component analysis of 2382 genesfrom normal airway transcriptome across 10 tissues. Principal componentanalysis (PCA) of 2382 genes from the normal airway transcriptome across10 different tissue types. Samples separate based on expression oftranscriptome genes.

FIG. 8 shows a hierarchical clustering of 51 genes across epithelialcell functional categories. Supervised hierarchical clustering of 51genes spanning mucin, dynein/microtubule, cytochrome P450, glutathione,and keratin functional gene categories. The 51 genes were clusteredacross the 10 tissue types separately for each functional group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes a novel method for prognosis anddiagnosis and follow-up for lung diseases. The method is based ondetecting gene expression changes of nose epithelial cells which we havediscovered closely mirror the gene expression changes in the lung.

Specifically, we have discovered that similar patterns of geneexpression changes can be found in the nose epithelial cells whencompared to lung epithelial changes in two model systems. In oneexperiment, we showed that a host gene expression in response to tobaccosmoke is similar whether it is measured from the lung epithelial cellsor from the nasal epithelial cells (FIG. 3). Accordingly, we havediscovered that we can rely on the results and data obtained withbronchial epithelial cells. This correlation is similar, typicallybetter than 75%, even if it is not identical. Thus, by looking at thesame gene groups that are diagnostic and/or prognostic for bronchialepithelial cells those groups are also diagnostic and/or prognostic fornasal epithelial cells. We also showed that gene expression changesdistinguishing between individuals affected with a lung diseases, suchas sarcoidosis, and from individuals not affected with that diseases.

Accordingly, the invention provides a substantially less invasive methodfor diagnosis, prognosis and follow-up of lung diseases using geneexpression analysis of samples from nasal epithelial cells.

One can take the nose epithelial cell sample from an individual using abrush or a swab. One can collect the nose epithelial cells in any wayknown to one skilled in the art. For example one can use nasal brushing.For example, one can collect the nasal epithelial cells by brushing theinferior turbinate and/or the adjacent lateral nasal wall. For example,following local anesthesia with 2% lidocaine solution, a CYROBRUSH®(MedScand Medical, Malmö, Sweden) or a similar device, is inserted intothe nare, for example the right nare, and under the inferior turbinateusing a nasal speculum for visualization. The brush is turned a coupleof times, for example 1, 2, 3, 4, 5 times, to collect epithelial cells.

To isolate nucleic acids from the cell sample, the cells can be placedimmediately into a solution that prevents nucleic acids fromdegradation. For example, if the cells are collected using theCYTOBRUSH, and one wishes to isolate RNA, the brush is placedimmediately into an RNA stabilizer solution, such as RNALATER®, AMBION®,Inc.

One can also isolate DNA. After brushing, the device can be placed in abuffer, such as phosphate buffered saline (PBS) for DNA isolation.

The nucleic acids are then subjected to gene expression analysis.Preferably, the nucleic acids are isolated and purified. However, if oneuses techniques such as microfluidic devises, cells may be placed intosuch device as whole cells without substantial purification.

In one preferred embodiment, one analyzes gene expression from nasalepithelial cells using gene/transcript groups and methods of using theexpression profile of these gene/transcript groups in diagnosis andprognosis of lung diseases.

We provide a method that is much less invasive than analysis ofbronchial samples. The method provided herein not only significantlyincreases the diagnostic accuracy of lung diseases, such as lung cancer,but also make the analysis much less invasive and thus much easier forthe patients and doctors to perform. When one combines the geneexpression analysis of the present invention with bronchoscopy, thediagnosis of lung cancer is dramatically better by detecting the cancerin an earlier stage than any other available method to date, and byproviding far fewer false negatives and/or false positives than anyother available method.

In one embodiment, one analyzes the nasal epithelial calls for a groupof gene transcripts that one can use individually and in groups orsubsets for enhanced diagnosis for lung diseases, such as lung cancer,using gene expression analysis.

On one embodiment, the invention provides a group of genes useful forlung disease diagnosis from a nasal epithelial cell sample as listed inTables 8, 9, and/or 10.

In one embodiment, one would analyze the nasal epithelial cells using atleast one and no more than 361 of the genes listed in Table 8. Forexample, one can analyze 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 10-15, 15-20,20-30, 30-40, 40-50, at least 10, at least 20, at least 30, at least 40at least 50, at least 60, at least 70, at least 80, at least 90, atleast 100, at least 110, at least 120, at least 130, at least 140, atleast 150, at least 160, at least or at maximum of 170, at least or atmaximum of 180, at least or at maximum of 190, at least or at maximum of200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,340, 350, 360, or at least 361 or at maximum of the 361 genes of genesas listed on Table 8.

In one embodiment, the invention provides genes

One example of the gene transcript groups useful in thediagnostic/prognostic tests of the invention is set forth in Table 6. Wehave found that taking any group that has at least 20 of the Table 6genes provides a much greater diagnostic capability than chance aloneand that these changes are substantially the same in the nasalepithelial cells than they are in the bronchial samples as described inPCT/US2006/014132.

Preferably one would analyze the nasal epithelial cells using more than20 of these gene transcript, for example about 20-100 and anycombination between, for example, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, and so on. Our preferred groups are the groups of 96 (Table 1), 84(Table 2), 50 (Table 3), 36 (Table 4), 80 (Table 5), 535 (Table 6) and20 (Table 7). In some instances, we have found that one can enhance theaccuracy of the diagnosis by adding additional genes to any of thesespecific groups.

Naturally, following the teachings of the present invention, one mayalso include one or more of the genes and/or transcripts presented inTables 1-7 into a kit or a system for a multicancer screening kit. Forexample, any one or more genes and or transcripts from Table 7 may beadded as a lung cancer marker for a gene expression analysis.

When one uses these groups, the genes in the group are compared to acontrol or a control group. The control groups can be non-smokers,smokers, or former smokers. Preferably, one compares the genetranscripts or their expression product in the nasal epithelial cellsample of an individual against a similar group, except that the membersof the control groups do not have the lung disorder, such as emphysemaor lung cancer. For example, comparing can be performed in the nasalepithelial cell sample from a smoker against a control group of smokerswho do not have lung cancer. When one compares the transcripts orexpression products against the control for increased expression ordecreased expression, which depends upon the particular gene and is setforth in the tables—not all the genes surveyed will show an increase ordecrease. However, at least 50% of the genes surveyed must provide thedescribed pattern. Greater reliability if obtained as the percentapproaches 100%. Thus, in one embodiment, one wants at least 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% of the genes surveyed toshow the altered pattern indicative of lung disease, such as lungcancer, as set forth in the tables as shown below.

The presently described gene expression profile can also be used toscreen for individuals who are susceptible for lung cancer. For example,a smoker, who is over a certain age, for example over 40 years old, or asmoker who has smoked, for example, a certain number of years, may wishto be screened for lung cancer. The gene expression analysis from nasalepithelial cells as described herein can provide an accurate very earlydiagnosis for lung cancer. This is particularly useful in diagnosis oflung cancer, because the earlier the cancer is detected, the better thesurvival rate is.

For example, when we analyzed the gene expression results, we found,that if one applies a less stringent threshold, the group of 80 genes aspresented in Table 5 are part of the most frequently chosen genes across1000 statistical test runs (see Examples below for more detailsregarding the statistical testing). Using random data, we have shownthat no random gene shows up more than 67 times out of 1000. Using sucha cutoff, the 535 genes of Table 6 in our data show up more than 67times out of 1000. All the 80 genes in Table 5 form a subset of the 535genes. Table 7 shows the top 20 genes which are subset of the 535 list.The direction of change in expression is shown using signal to noiseratio. A negative number in Tables 5, 6, and 7 means that expression ofthis gene or transcript is up in lung cancer samples. Positive number inTable 5, 6, and 7, indicates that the expression of this gene ortranscript is down in lung cancer.

Accordingly, any combination of the genes and/or transcripts of Table 6can be used. In one embodiment, any combination of at least 5-10, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80, 80-90, 90-100, 100-120,120-140, 140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210,210-220, 220-230, 230-240, 240-250, 250-260, 260-270, 270-280, 280-290,290-300, 300-310, 310-320, 320-330, 330-340, 340-350, 350-360, 360-370,370-380, 380-390, 390-400, 400-410, 410-420, 420-430, 430-440, 440-450,450-460, 460-470, 470-480, 480-490, 490-500, 500-510, 510-520, 520-530,and up to about 535 genes selected from the group consisting of genes ortranscripts as shown in the Table 6.

Table 7 provides 20 of the most frequently variably expressed genes inlung cancer when compared to samples without cancer. Accordingly, in oneembodiment, any combination of about 3-5, 5-10, 11, 12, 13, 14, 15, 16,17, 18, 19, or all 20 genes and/or transcripts of Table 7, or anysub-combination thereof are used.

In one embodiment, the invention provides a gene group the expressionprofile of nasal epithelial cells which is useful in diagnosing lungdiseases and which comprises probes that hybridize ranging from 1 to 96and all combinations in between for example 5, 10, 15, 20, 25, 30, 35,at least about 36, at least to 40, at lest to 50, at least to 60, to atleast 70, to at least 80, to at least 90, or all of the following 96gene sequences: NM_(—)003335; NM_(—)000918; NM_(—)006430.1;NM_(—)001416.1; NM_(—)004090; NM_(—)006406.1; NM_(—)003001.2;NM_(—)001319; NM_(—)006545.1; NM_(—)021145.1; NM_(—)002437.1;NM_(—)006286; NM_(—)001003698//NM_(—)001003699///NM_(—)002955;NM_(—)001123///NM_(—)006721; NM_(—)024824; NM_(—)004935.1;NM_(—)002853.1; NM_(—)019067.1; NM_(—)024917.1; NM_(—)020979.1;NM_(—)005597.1; NM_(—)007031.1; NM_(—)0095901.; NM_(—)020217.1;NM_(—)025026.1; NM_(—)014709.1; NM_(—)014896.1; AF010144;NM_(—)005374.1; NM_(—)001696; NM_(—)005494///NM_(—)058246;NM_(—)006534///NM_(—)181659; NM_(—)006368; NM_(—)002268///NM_(—)032771;NM_(—)014033; NM_(—)016138; NM_(—)007048///NM_(—)194441; NM_(—)006694;NM_(—)000051///NM_(—)138292///NM_(—)138293;NM_(—)000410///NM_(—)139002///NM_(—)139003///NM_(—)139004///NM_(—)139005///NM_(—)139006///NM_(—)139007///NM_(—)139008///NM_(—)139009///NM_(—)139010///NM_(—)139011;NM_(—)004691; NM_(—)012070///NM_(—)139321///NM_(—)139322; NM_(—)006095;A1632181; AW024467, NM_(—)021814; NM_(—)005547.1; NM_(—)203458;NM_(—)015547///NM_(—)147161; AB007958.1; NM 207488;NM_(—)005809///NM_(—)181737///NM_(—)181738; NM_(—)016248///NM_(—)144490;AK022213.1; NM_(—)005708; NM_(—)207102; AK023895;NM_(—)144606///NM_(—)144997; NM_(—)018530; AK021474; U43604.1; AU147017;AF222691.1; NM_(—)015116;NM_(—)001005375///NM_(—)001005785///NM_(—)001005786///NM_(—)004081///NM_(—)020363///NM_(—)020364///NM_(—)020420;AC004692; NM_(—)001014; NM_(—)000585///NM_(—)172174///NM_(—)172175;NM_(—)054020///NM_(—)172095///NM_(—)172096///NM_(—)172097; BE466926;NM_(—)018011; NM_(—)024077; NM_(—)012394;NM_(—)0190111///NM_(—)207111///NM_(—)207116; NM_(—)017646; NM_(—)021800;NM_(—)016049; NM_(—)014395; NM_(—)014336; NM_(—)018097; NM_(—)019014;NM_(—)024804; NM_(—)018260; NM_(—)018118; NM_(—)014128; NM_(—)024084;NM_(—)005294; AF077053; NM_(—)138387; NM_(—)024531, NM_(—)000693;NM_(—)018509; NM_(—)033128; NM_(—)020706; A1523613; and NM_(—)014884

In one embodiment, the invention provides a gene group the expressionprofile of nasal epithelial cells of which is useful in diagnosing lungdiseases and comprises probes that hybridize to at least, for example,5, 10, 15, 20, 25, 30, 35, at least about 36, at least to 40, at leastto 50, at least to 60, to at least 70, to at least 80, to all of thefollowing 84 gene sequences: NM_(—)030757.1; R83000; AK021571.1;NM_(—)014182.1; NM_(—)17932.1; U85430.1; AI683552; BC002642.1; AW024467;NM_(—)030972.1; BC021135.1; AL161952.1; AK026565.1; AK023783.1;BF218804; NM_(—)0012811; NM_(—)024006.1; AK023843.1; BC001602.1;BC034707.1; BC064619.1; AY280502.1; BC059387.1; AF135421.1; BC061522.1;L76200.1; U50532.1; BC006547.2; BC008797.2; BC000807.1; AL080112.1;BC033718.1///BC046176.1///BC038443.1; NM_(—)000346.1; BC008710.1;Hs.288575 (UNIGENE ID); AF020591.1; BC000423.2; BC002503.2; BC008710.1;BC009185.2; Hs.528304 (UNIGENE ID); U50532.1; BC013923.2; BC031091;NM_(—)007062; Hs.249591 (Unigene ID); BC075839.1///BC073760.1;BC072436.1///BC004560.2; BC001016.2; Hs.286261 (Unigene ID); AF348514.1;BC005023.1; BC066337.1///BC058736.1///BC050555.1; Hs.216623 (UnigeneID); BC072400.1; BC041073.1; U43965.1; BC021258.2; BC016057.1;BC016713.1///BC014535.1///AF237771.1; BC000360.2; BC007455.2;BC000701.2; BC010067.2; BC023528.2///; BC047680.1; BC064957.1; Hs.156701(Unigene ID); BC030619.2; BC008710.1; U43965.1; BC066329.1; Hs.438867(Unigene ID); BC035025.2///BC050330.1; BC023976.2;BC074852.2///BC074851.2; Hs.445885 (Unigene ID);BC008591.2///BC050440.1///; BC048096.1; AF365931.1; AF257099.1; andBC028912.1.

In one embodiment, the invention provides a gene group the expressionprofile of nasal epithelial cells which is useful in diagnosing lungdiseases and comprises probes that hybridize to at least, for example 5,10, 15, 20, 25, 30, preferably at least about 36, still more preferablyat least to 40, still more preferably at lest to 45, still morepreferably all of the following 50 gene sequences, although it caninclude any and all members, for example, 20, 21, 22, up to andincluding 36: NM_(—)007062.1; NM_(—)001281.1; BC000120.1;NM_(—)014255.1; BC002642.1; NM_(—)000346.1; NM_(—)006545.1; BG034328;NM_(—)021822.1; NM_(—)021069.1; NM_(—)019067.1; NM_(—)017925.1;NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1; AF126181.1; U93240.1;U90552.1; AF151056.1; U85430.1; U51007.1; BC005969.1; NM_(—)002271.1;AL566172; AB014576.1; BF218804; AK022494.1; AA114843; BE467941;NM_(—)003541.1; R83000; AL161952.1; AK023843.1; AK021571.1; AK023783.1;AU147182; AL080112.1; AW971983; AI683552; NM_(—)024006.1; AK026565.1;NM_(—)014182.1; NM_(—)021800.1; NM_(—)016049.1; NM_(—)019023.1;NM_(—)021971.1; NM_(—)014128.1; AK025651.1; AA133341; and AF198444.1. Inone preferred embodiment, one can use at least 20-30, 30-40, of the 50genes that overlap with the individual predictor genes identified in theanalysis using the t-test, and, for example, 5-9 of the non-overlappinggenes, identified using the t-test analysis as individual predictorgenes, and combinations thereof.

In one embodiment, the invention provides a gene group the expressionprofile of nasal epithelial cells which is useful in diagnosing lungdiseases and comprises probes that hybridize to at least for example 5,10, 15, 20, preferably at least about 25, still more preferably at leastto 30, still more preferably all of the following 36 gene sequences:NM_(—)007062.1; NM_(—)001281.1; BC002642.1; NM_(—)000346.1;NM_(—)006545.1; BG034328; NM_(—)019067.1; NM_(—)017925.1;NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1;NM_(—)002268///NM_(—)032771; NM_(—)007048///NM_(—)194441; NM_(—)006694;U85430.1; NM_(—)004691; AB014576.1; BF218804; BE467941; R83000;AL161952.1; AK023843.1; AK021571.1; AK023783.1; AL080112.1; AW971983;AI683552; NM_(—)024006.1; AK026565.1; NM_(—)014182.1; NM_(—)021800.1;NM_(—)016049.1; NM_(—)021971.1; NM_(—)014128.1; AA133341; andAF198444.1. In one preferred embodiment, one can use at least 20 of the36 genes that overlap with the individual predictors and, for example,5-9 of the non-overlapping genes, and combinations thereof.

The expression of the gene groups in an individual sample can beanalyzed using any probe specific to the nucleic acid sequences orprotein product sequences encoded by the gene group members. Forexample, in one embodiment, a probe set useful in the methods of thepresent invention is selected from the nucleic acid probes of between10-15, 15-20, 20-180, preferably between 30-180, still more preferablybetween 36-96, still more preferably between 36-84, still morepreferably between 36-50 probes, included in the Affymetrix Inc. genechip of the Human Genome U133 Set and identified as probe ID Nos:208082_x_at, 214800_x_at, 215208_x_at, 218556_at, 207730_x_at,210556_at, 217679_x_at, 202901_x_at, 213939_s_at, 208137_x_at,214705_at, 215001_s_at, 218155_x_at, 215604_x_at, 212297_at,201804_x_at, 217949_s_at, 215179_x_at, 211316_x_at, 217653_x_at,266_s_at, 204718_at 211916_s_at, 215032_at, 219920_s_at, 211996_s_at,200075_s_at, 214753_at, 204102_s_at, 202419_at, 214715_x_at,216859_x_at, 215529_x_at, 202936_s_at, 212130_x_at, 215204 at,218735_s_at, 200078_s_at, 203455_s_at, 212227_x_at, 222282_at,219678_x_at, 208268_at, 221899_at, 213721_at, 214718_at, 201608_s_at,205684_s_at, 209008_x_at, 200825_s_at, 218160_at, 57739_at, 211921_x_at,218074_at, 200914_x_at, 216384_x_at, 214594_x_at, 222122_s_at,204060_s_at, 215314_at, 208238_x_at, 210705_s_at, 211184_s_at,215418_at, 209393_s_at, 210101_x_at, 212052_s_at, 215011_at,221932_s_at, 201239_s_at, 215553_x_at, 213351_s_at, 202021_x_at,209442_x_at, 210131_x_at, 217713_x_at, 214707_x_at, 203272_s_at,206279_at, 214912_at, 201729_s_at, 205917_at, 200772_x_at, 202842_s_at,203588_s_at, 209703_x_at, 217313_at, 217588_at, 214153_at, 222155_s_at,203704_s_at, 220934_s_at, 206929_s_at, 220459_at, 215645_at, 217336_at,203301_s_at, 207283_at, 222168_at, 222272_x_at, 219290_x_at,204119_s_at, 215387_x_at, 222358_x_at, 205010_at, 1316_at, 216187_x_at,208678_at, 222310_at, 210434_x_at, 220242_x_at, 207287_at, 207953_at,209015_s_at, 221759_at, 220856_x_at, 200654_at, 220071_x_at,216745_x_at, 218976_at, 214833_at, 202004_x_at, 209653_at, 210858_x_at,212041_at, 221294_at, 207020_at, 204461_x_at, 205367_at, 219203_at,215067_x_at, 212517_at, 220215_at, 201923_at, 215609_at, 207984_s_at,215373_x_at, 216110_x_at, 215600_x_at, 216922_x_at, 215892_at,201530_x_at, 217371_s_at, 222231_s_at, 218265_at, 201537_s_at,221616_s_at, 213106_at, 215336_at, 209770_at, 209061_at, 202573_at,207064_s_at, 64371_at, 219977_at, 218617_at, 214902_x_at, 207436_x_at,215659_at, 204216_s_at, 214763_at, 200877_at, 218425_at, 203246_s_at,203466_at, 204247_s_at, 216012_at, 211328_x_at, 218336_at, 209746_s_at,214722_at, 214599_at, 220113_x_at, 213212_x_at, 217671_at, 207365_x_at,218067_s_at, 205238_at, 209432_s_at, and 213919_at. In one preferredembodiment, one can use at least, for example, 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 110, 120, 130, 140, 150, 160,or 170 of the 180 genes that overlap with the individual predictorsgenes and, for example, 5-9 of the non-overlapping genes andcombinations thereof.

Sequences for the Affymetrix probes are available from Affymetrix. Otherprobes and sequences that recognize the genes of interest can be easilyprepared using, e.g. synthetic oligonucleotides recombinantoligonucleotides. These sequences can be selected from any, preferablyunique part of the gene based on the sequence information publiclyavailable for the genes that are indicated by their HUGO ID, GenBank No.or Unigene No.

One can analyze the expression data to identify expression pattersassociated with any lung disease. For example, one can analyze diseasescaused by exposure to air pollutants, such as cigarette smoke, asbestosor any other pollutant. For example, the analysis can be performed asfollows. One first scans a gene chip or mixture of beads comprisingprobes that are hybridized with a study group samples. For example, onecan use samples of non-smokers and smokers, non-asbestos exposedindividuals and asbestos-exposed individuals, non-smog exposedindividuals and smog-exposed individuals, smokers without a lung diseaseand smokers with lung disease, to obtain the differentially expressedgene groups between individuals with no lung disease and individualswith lung disease. One must, of course select appropriate groups,wherein only one air pollutant can be selected as a variable. So, forexample, one can compare non-smokers exposed to asbestos but not smogand non-smokers not exposed to asbestos or smog.

The obtained expression analysis, such as microarray or microbead rawdata consists of signal strength and detection p-value. One normalizesor scales the data, and filters the poor quality chips/bead sets basedon images of the expression data, control probes, and histograms. Onealso filters contaminated specimens which contain non-epithelial cells.Lastly, one filters the genes of importance using detection p-value.This results in identification of transcripts present in normal airways(normal airway transcriptome). Variability and multiple regressionanalysis can be used. This also results in identification of effects ofsmoking on airway epithelial cell transcription. For this analysis, onecan use T-test and Pearson correlation analysis. One can also identify agroup or a set of transcripts that are differentially expressed insamples with lung disease, such as lung cancer and samples withoutcancer. This analysis was performed using class prediction models.

For analysis of the data, one can use, for example, a weighted votingmethod. The weighted voting method ranks, and gives a weight “p” to allgenes by the signal to noise ration of gene expression between twoclasses:P=mean_((class 1))−mean_((class 2))/sd_((class 1))=sd_((class 2)).Committees of variable sizes of the top ranked genes are used toevaluate test samples, but genes with more significant p-values can bemore heavily weighed. Each committee genes in test sample votes for oneclass or the other, based on how close that gene expression level is tothe class 1 mean or the class 2 mean. V_((gene A))=P_((gene A)), i.e.level of expression in test sample less the average of the meanexpression values in the two classes. Votes for each class are talliedand the winning class is determined along with prediction strength asPS=V_(win)−V_(lose)/V_(win)+V_(lose). Finally, the accuracy can bevalidated using cross-validation+/−independent samples.

Table 1 shows 96 genes that were identified as a group distinguishingsmokers with cancer from smokers without cancer. The difference inexpression is indicated at the column on the right as either “down”,which indicates that the expression of that particular transcript waslower in smokers with cancer than in smokers without cancer, and “up”,which indicates that the expression of that particular transcript washigher in smokers with cancer than smokers without cancer. In oneembodiment, the exemplary probes shown in the column “Affymetrix Id inthe Human Genome U133 chip” can be used.

TABLE 1 96 Gene Group Affymetrix ID for an Expression example in cancerprobe compared to identifying a sample the gene GenBank ID Gene Namewith no cancer. 1316_at NM_003335 UBE1L down 200654_at NM_000918 P4HB up200877_at NM_006430.1 CCT4 up 201530_x_at NM_001416.1 EIF4A1 up201537_s_at NM_004090 DUSP3 up 201923_at NM_006406.1 PRDX4 up202004_x_at NM_003001.2 SDHC up 202573_at NM_001319 CSNK1G2 down203246_s_at NM_006545.1 TUSC4 up 203301_s_at NM_021145.1 DMTF1 down203466_at NM_002437.1 MPV17 up 203588_s_at NM_006286 TFDP2 up203704_s_at NM_001003698 /// RREB1 down NM_001003699 /// NM_002955204119_s_at NM_001123 /// ADK up NM_006721 204216_s_at NM_024824FLJ11806 up 204247_s_at NM_004935.1 CDK5 up 204461_x_at NM_002853.1 RAD1down 205010_at NM_019067.1 FLJ10613 down 205238_at NM_024917.1 CXorf34down 205367_at NM_020979.1 APS down 206929_s_at NM_005597.1 NFIC down207020_at NM_007031.1 HSF2BP down 207064_s_at NM_009590.1 AOC2 down207283_at NM_020217.1 DKFZp547I014 down 207287_at NM_025026.1 FLJ14107down 207365_x_at NM_014709.1 USP34 down 207436_x_at NM_014896.1 KIAA0894down 207953_at AF010144 — down 207984_s_at NM_005374.1 MPP2 down208678_at NM_001696 ATP6V1E1 up 209015_s_at NM_005494 /// DNAJB6 upNM_058246 209061_at NM_006534 /// NCOA3 down NM_181659 209432_s_atNM_006368 CREB3 up 209653_at NM_002268 /// KPNA4 up NM_032771209703_x_at NM_014033 DKFZP586A0522 down 209746_s_at NM_016138 COQ7 down209770_at NM_007048 /// BTN3A1 down NM_194441 210434_x_at NM_006694 JTBup 210858_x_at NM_000051 /// ATM down NM_138292 /// NM_138293211328_x_at NM_000410 /// HFE down NM_139002 /// NM_139003 /// NM_139004/// NM_139005 /// NM_139006 /// NM_139007 /// NM_139008 /// NM_139009/// NM_139010 /// NM_139011 212041_at NM_004691 ATP6V0D1 up 212517_atNM_012070 /// ATRN down NM_139321 /// NM_139322 213106_at NM_006095ATP8A1 down 213212_x_at AI632181 — down 213919_at AW024467 — down214153_at NM_021814 ELOVL5 down 214599_at NM_005547.1 IVL down 214722_atNM_203458 N2N down 214763_at NM_015547 /// THEA down NM_147161 214833_atAB007958.1 KIAA0792 down 214902_x_at NM_207488 FLJ42393 down 215067_x_atNM_005809 /// PRDX2 down NM_181737 /// NM_181738 215336_at NM_016248 ///AKAP11 down NM_144490 215373_x_at AK022213.1 FLJ12151 down 215387_x_atNM_005708 GPC6 down 215600_x_at NM_207102 FBXW12 down 215609_at AK023895— down 215645_at NM_144606 /// FLCN down NM_144997 215659_at NM_018530GSDML down 215892_at AK021474 — down 216012_at U43604.1 — down216110_x_at AU147017 — down 216187_x_at AF222691.1 LNX1 down 216745_x_atNM_015116 LRCH1 down 216922_x_at NM_001005375 /// DAZ2 down NM_001005785/// NM_001005786 /// NM_004081 /// NM_020363 /// NM_020364 /// NM_020420217313_at AC004692 — down 217336_at NM_001014 RPS10 down 217371_s_atNM_000585 /// IL15 down NM_172174 /// NM_172175 217588_at NM_054020 ///CATSPER2 down NM_172095 /// NM_172096 /// NM_172097 217671_at BE466926 —down 218067_s_at NM_018011 FLJ10154 down 218265_at NM_024077 SECISBP2down 218336_at NM_012394 PFDN2 up 218425_at NM_019011 /// TRIAD3 downNM_207111 /// NM_207116 218617_at NM_017646 TRIT1 down 218976_atNM_021800 DNAJC12 up 219203_at NM_016049 C14orf122 up 219290_x_atNM_014395 DAPP1 down 219977_at NM_014336 AIPL1 down 220071_x_atNM_018097 C15orf25 down 220113_x_at NM_019014 POLR1B down 220215_atNM_024804 FLJ12606 down 220242_x_at NM_018260 FLJ10891 down 220459_atNM_018118 MCM3APAS down 220856_x_at NM_014128 down 220934_s_at NM_024084MGC3196 down 221294_at NM_005294 GPR21 down 221616_s_at AF077053 PGK1down 221759_at NM_138387 G6PC3 up 222155_s_at NM_024531 GPR172A up222168_at NM_000693 ALDH1A3 down 222231_s_at NM_018509 PRO1855 up222272_x_at NM_033128 SCIN down 222310_at NM_020706 SFRS15 down222358_x_at AI523613 — down 64371_at NM_014884 SFRS14 down

Table 2 shows one preferred 84 gene group that has been identified as agroup distinguishing smokers with cancer from smokers without cancer.The difference in expression is indicated at the column on the right aseither “down”, which indicates that the expression of that particulartranscript was lower in smokers with cancer than in smokers withoutcancer, and “up”, which indicates that the expression of that particulartranscript was higher in smokers with cancer than smokers withoutcancer. These genes were identified using traditional Student's t-testanalysis.

In one embodiment, the exemplary probes shown in the column “AffymetrixId in the Human Genome U133 chip” can be used in the expressionanalysis.

TABLE 2 84 Gene Group Direction in GenBank ID Cancer (unless compared toa otherwise Gene Name non-cancer mentioned) Abbreviation sampleAffymetrix ID NM_030757.1 MKRN4 down 208082_x_at R83000 BTF3 down214800_x_at AK021571.1 MUC20 down 215208_x_at NM_014182.1 ORMDL2 up218556_at NM_17932.1 FLJ20700 down 207730_x_at U85430.1 NFATC3 down210556_at AI683552 — down 217679_x_at BC002642.1 CTSS down 202901_x_atAW024467 RIPX down 213939_s_at NM_030972.1 MGC5384 down 208137_x_atBC021135.1 INADL down 214705_at AL161952.1 GLUL down 215001_s_atAK026565.1 FLJ10534 down 218155_x_at AK023783.1 — down 215604_x_atBF218804 AFURS1 down 212297_at NM_001281.1 CKAP1 up 201804_x_atNM_024006.1 IMAGE3455200 up 217949_s_at AK023843.1 PGF down 215179_x_atBC001602.1 CFLAR down 211316_x_at BC034707.1 — down 217653_x_atBC064619.1 CD24 down 266_s_at AY280502.1 EPHB6 down 204718_at BC059387.1MYO1A down 211916_s_at — down 215032_at AF135421.1 GMPPB up 219920_s_atBC061522.1 MGC70907 down 211996_s_at L76200.1 GUK1 up 200075_s_atU50532.1 CG005 down 214753_at BC006547.2 EEF2 down 204102_s_atBC008797.2 FVT1 down 202419_at BC000807.1 ZNF160 down 214715_x_atAL080112.1 — down 216859_x_at BC033718.1 /// C21orf106 down 215529_x_atBC046176.1 /// BC038443.1 NM_000346.1 SOX9 up 202936_s_at BC008710.1SUI1 up 212130_x_at Hs.288575 — down 215204_at (Unigene ID) AF020591.1AF020591 down 218735_s_at BC000423.2 ATP6V0B up 200078_s_at BC002503.2SAT down 203455_s_at BC008710.1 SUI1 up 212227_x_at — down 222282_atBC009185.2 DCLRE1C down 219678_x_at Hs.528304 ADAM28 down 208268_at(UNIGENE ID) U50532.1 CG005 down 221899_at BC013923.2 SOX2 down213721_at BC031091 ODAG down 214718_at NM_007062 PWP1 up 201608_s_atHs.249591 FLJ20686 down 205684_s_at (Unigene ID) BC075839.1 /// KRT8 up209008_x_at BC073760.1 BC072436.1 /// HYOU1 up 200825_s_at BC004560.2BC001016.2 NDUFA8 up 218160_at Hs.286261 FLJ20195 down 57739_at (UnigeneID) AF348514.1 — down 211921_x_at BC005023.1 CGI-128 up 218074_atBC066337.1 /// KTN1 down 200914_x_at BC058736.1 /// BC050555.1 — down216384_x_at Hs.216623 ATP8B1 down 214594_x_at (Unigene ID) BC072400.1THOC2 down 222122_s_at BC041073.1 PRKX down 204060_s_at U43965.1 ANK3down 215314_at — down 208238_x_at BC021258.2 TRIM5 down 210705_s_atBC016057.1 USH1C down 211184_s_at BC016713.1 /// PARVA down 215418_atBC014535.1 /// AF237771.1 BC000360.2 EIF4EL3 up 209393_s_at BC007455.2SH3GLB1 up 210101_x_at BC000701.2 KIAA0676 down 212052_s_at BC010067.2CHC1 down 215011_at BC023528.2 /// C14orf87 up 221932_s_at BC047680.1BC064957.1 KIAA0102 up 201239_s_at Hs.156701 — down 215553_x_at (UnigeneID) BC030619.2 KIAA0779 down 213351_s_at BC008710.1 SUI1 up 202021_x_atU43965.1 ANK3 down 209442_x_at BC066329.1 SDHC up 210131_x_at Hs.438867— down 217713_x_at (Unigene ID) BC035025.2 /// ALMS1 down 214707_x_atBC050330.1 BC023976.2 PDAP2 up 203272_s_at BC074852.2 /// PRKY down206279_at BC074851.2 Hs.445885 KIAA1217 down 214912_at (Unigene ID)BC008591.2 /// KIAA0100 up 201729_s_at BC050440.1 /// BC048096.1AF365931.1 ZNF264 down 205917_at AF257099.1 PTMA down 200772_x_atBC028912.1 DNAJB9 up 202842_s_at

Table 3 shows one preferred 50 gene group that was identified as a groupdistinguishing smokers with cancer from smokers without cancer. Thedifference in expression is indicated at the column on the right aseither “down”, which indicates that the expression of that particulartranscript was lower in smokers with cancer than in smokers withoutcancer, and “up”, which indicates that the expression of that particulartranscript was higher in smokers with cancer than smokers withoutcancer.

This gene group was identified using the GenePattern server from theBroad Institute, which includes the Weighted Voting algorithm. Thedefault settings, i.e., the signal to noise ratio and no gene filtering,were used.

In one embodiment, the exemplary probes shown in the column “AffymetrixId in the Human Genome U133 chip” can be used in the expressionanalysis.

TABLE 3 50 Gene Group Direction GenBank ID Gene Name in CancerAffymetrix ID NM_007062.1 PWP1 up in cancer 201608_s_at NM_001281.1CKAP1 up in cancer 201804_x_at BC000120.1 up in cancer 202355_s_atNM_014255.1 TMEM4 up in cancer 202857_at BC002642.1 CTSS up in cancer202901_x_at NM_000346.1 SOX9 up in cancer 202936_s_at NM_006545.1 NPR2Lup in cancer 203246_s_at BG034328 up in cancer 203588_s_at NM_021822.1APOBEC3G up in cancer 204205_at NM_021069.1 ARGBP2 up in cancer204288_s_at NM_019067.1 FLJ10613 up in cancer 205010_at NM_017925.1FLJ20686 up in cancer 205684_s_at NM_017932.1 FLJ20700 up in cancer207730_x_at NM_030757.1 MKRN4 up in cancer 208082_x_at NM_030972.1MGC5384 up in cancer 208137_x_at AF126181.1 BCG1 up in cancer208682_s_at U93240.1 up in cancer 209653_at U90552.1 up in cancer209770_at AF151056.1 up in cancer 210434_x_at U85430.1 NFATC3 up incancer 210556_at U51007.1 up in cancer 211609_x_at BC005969.1 up incancer 211759_x_at NM_002271.1 up in cancer 211954_s_at AL566172 up incancer 212041_at AB014576.1 KIAA0676 up in cancer 212052_s_at BF218804AFURS1 down in cancer 212297_at AK022494.1 down in cancer 212932_atAA114843 down in cancer 213884_s_at BE467941 down in cancer 214153_atNM_003541.1 HIST1H4K down in cancer 214463_x_at R83000 BTF3 down incancer 214800_x_at AL161952.1 GLUL down in cancer 215001_s_at AK023843.1PGF down in cancer 215179_x_at AK021571.1 MUC20 down in cancer215208_x_at AK023783.1 — down in cancer 215604_x_at AU147182 down incancer 215620_at AL080112.1 — down in cancer 216859_x_at AW971983 downin cancer 217588_at AI683552 — down in cancer 217679_x_at NM_024006.1IMAGE3455200 down in cancer 217949_s_at AK026565.1 FLJ10534 down incancer 218155_x_at NM_014182.1 ORMDL2 down in cancer 218556_atNM_021800.1 DNAJC12 down in cancer 218976_at NM_016049.1 CGI-112 down incancer 219203_at NM_019023.1 PRMT7 down in cancer 219408_at NM_021971.1GMPPB down in cancer 219920_s_at NM_014128.1 — down in cancer220856_x_at AK025651.1 down in cancer 221648_s_at AA133341 C14orf87 downin cancer 221932_s_at AF198444.1 down in cancer 222168_at

Table 4 shows one preferred 36 gene group that was identified as a groupdistinguishing smokers with cancer from smokers without cancer. Thedifference in expression is indicated at the column on the right aseither “down”, which indicates that the expression of that particulartranscript was lower in smokers with cancer than in smokers withoutcancer, and “up”, which indicates that the expression of that particulartranscript was higher in smokers with cancer than smokers withoutcancer.

In one embodiment, the exemplary probes shown in the column “AffymetrixId in the Human Genome U133 chip” can be used in the expressionanalysis.

TABLE 4 36 Gene Group GenBank ID Gene Name Affymetrix ID NM_007062.1PWP1 201608_s_at NM_001281.1 CKAP1 201804_x_at BC002642.1 CTSS202901_x_at NM_000346.1 SOX9 202936_s_at NM_006545.1 NPR2L 203246_s_atBG034328 203588_s_at NM_019067.1 FLJ10613 205010_at NM_017925.1 FLJ20686205684_s_at NM_017932.1 FLJ20700 207730_x_at NM_030757.1 MKRN4208082_x_at NM_030972.1 MGC5384 208137_x_at NM_002268 /// NM_032771KPNA4 209653_at NM_007048 /// NM_194441 BTN3A1 209770_at NM_006694 JBT210434_x_at U85430.1 NFATC3 210556_at NM_004691 ATP6V0D1 212041_atAB014576.1 KIAA0676 212052_s_at BF218804 AFURS1 212297_at BE467941214153_at R83000 BTF3 214800_x_at AL161952.1 GLUL 215001_s_at AK023843.1PGF 215179_x_at AK021571.1 MUC20 215208_x_at AK023783.1 — 215604_x_atAL080112.1 — 216859_x_at AW971983 217588_at AI683552 — 217679_x_atNM_024006.1 IMAGE3455200 217949_s_at AK026565.1 FLJ10534 218155_x_atNM_014182.1 ORMDL2 218556_at NM_021800.1 DNAJC12 218976_at NM_016049.1CGI-112 219203_at NM_021971.1 GMPPB 219920_s_at NM_014128.1 —220856_x_at AA133341 C14orf87 221932_s_at AF198444.1 222168_at

In one embodiment, the gene group of the present invention comprises atleast, for example, 5, 10, 15, 20, 25, 30, more preferably at least 36,still more preferably at least about 40, still more preferably at leastabout 50, still more preferably at least about 60, still more preferablyat least about 70, still more preferably at least about 80, still morepreferably at least about 86, still more preferably at least about 90,still more preferably at least about 96 of the genes as shown in Tables1-4.

In one preferred embodiment, the gene group comprises 36-180 genesselected from the group consisting of the genes listed in Tables 1-4.

In one embodiment, the invention provides group of genes the expressionof which is lower in individuals with cancer.

Accordingly, in one embodiment, the invention provides of a group ofgenes useful in diagnosing lung diseases, wherein the expression of thegroup of genes is lower in individuals exposed to air pollutants withcancer as compared to individuals exposed to the same air pollutant whodo not have cancer, the group comprising probes that hybridize at least5, preferably at least about 5-10, still more preferably at least about10-20, still more preferably at least about 20-30, still more preferablyat least about 30-40, still more preferably at least about 40-50, stillmore preferably at least about 50-60, still more preferably at leastabout 60-70, still more preferably about 72 genes consisting oftranscripts (transcripts are identified using their Genbank ID orUnigene ID numbers and the corresponding gene names appear in Table 1):NM_(—)003335; NM_(—)001319; NM_(—)021145.1;NM_(—)001003698///NM_(—)001003699///; NM_(—)002955; NM_(—)002853.1;NM_(—)019067.1; NM_(—)024917.1; NM_(—)020979.1; NM_(—)005597.1;NM_(—)007031.1; NM_(—)009590.1; NM_(—)020217.1; NM_(—)025026.1;NM_(—)014709.1; NM_(—)014896.1; AF010144; NM_(—)005374.1;NM_(—)006534///NM_(—)181659; NM_(—)014033; NM_(—)016138;NM_(—)007048///NM_(—)194441; NM_(—)000051///NM_(—)138292///NM_(—)138293;NM_(—)000410///NM_(—)139002///NM_(—)139003///NM_(—)139004///NM_(—)139005///NM_(—)139006///NM_(—)139007///NM_(—)139008//NM_(—)139009///NM_(—)139010///NM_(—)139011;NM_(—)012070///NM_(—)139321///NM_(—)139322; NM_(—)006095; AI632181;AW024467; NM_(—)021814; NM_(—)005547.1; NM 203458;NM_(—)015547///NM_(—)147161; AB007958.1; NM_(—)207488;NM_(—)005809///NM_(—)181737///NM_(—)181738; NM_(—)016248///NM_(—)144490;AK022213.1; NM_(—)005708; NM_(—)207102; AK023895;NM_(—)144606///NM_(—)144997; NM_(—)018530; AK021474; U43604.1; AU147017;AF222691.1; NM_(—)015116;NM_(—)001005375///NM_(—)001005785///NM_(—)001005786///NM_(—)004081///NM_(—)020363///NM_(—)020364///NM_(—)020420;AC004692; NM_(—)001014; NM_(—)000585///NM_(—)172174///NM_(—)72175;NM_(—)054020///NM_(—)172095///NM_(—)172096///NM_(—)172097; BE466926;NM_(—)018011; NM_(—)024077; NM_(—)019011///NM_(—)20711///NM_(—)207116;NM_(—)017646; NM_(—)014395; NM_(—)014336; NM_(—)018097; NM_(—)019014;NM_(—)024804; NM_(—)018260; NM_(—)018118; NM_(—)014128, NM_(—)024084;NM_(—)005294; AF077053; NM_(—)000693; NM_(—)033128; NM_(—)020706;AI523613; and NM_(—)014884.

In another embodiment, the invention provides of a group of genes usefulin diagnosing lung diseases wherein the expression of the group of genesis lower in individuals exposed to air pollutants with cancer ascompared to individuals exposed to the same air pollutant who do nothave cancer, the group comprising probes that hybridize at least 5,preferably at least about 5-10, still more preferably at least about10-20, still more preferably at least about 20-30, still more preferablyat least about 3040, still more preferably at least about 40-50, stillmore preferably at least about 50-60, still more preferably about 63genes consisting of transcripts (transcripts are identified using theirGenBank ID or Unigene ID numbers and the corresponding gene names appearin Table 2): NM_(—)030757.1; R83000; AK021571.1; NM_(—)17932.1;U85430.1; A1683552; BC002642.1; AW024467; NM_(—)030972.1; BC021135.1;AL161952.1; AK026565.1; AK023783.1; BF218804; AK023843.1; BC001602.1;BC034707.1; BC064619.1; AY280502.1; BC059387.1; BC061522.1; U50532.1;BC006547.2; BC008797.2; BC000807.1; AL080112.1;BC033718.1///BC046176.1///; BC038443.1; Hs.288575 (UNIGENE ID);AF020591.1; BC002503.2; BC009185.2; Hs.528304 (UNIGENE ID); U50532.1;BC013923.2; BC031091; Hs.249591 (Unigene ID); Hs.286261 (Unigene ID);AF348514.1; BC066337.1///BC058736.1///BC050555.1; Hs.216623 (UnigeneID); BC072400.1; BC041073.1; U43965.1; BC021258.2; BC016057.1;BC016713.1///BC014535.1///AF237771.1; BC000701.2; BC010067.2; Hs.156701(Unigene ID); BC030619.2; U43965.1; Hs.438867 (Unigene ID);BC035025.2///BC050330.1; BC074852.2///BC074851.2; Hs.445885 (UnigeneID); AF365931.1; and AF257099.1

In another embodiment, the invention provides of a group of genes usefulin diagnosing lung diseases wherein the expression of the group of genesis lower in individuals exposed to air pollutants with cancer ascompared to individuals exposed to the same air pollutant who do nothave cancer, the group comprising probes that hybridize at least 5,preferably at least about 5-10, still more preferably at least about10-20, still more preferably at least about 20-25, still more preferablyabout 25 genes consisting of transcripts (transcripts are identifiedusing their GenBank ID or Unigene ID numbers and the corresponding genenames appear in Table 3): BF218804; AK022494.1; AA114843; BE467941;NM_(—)003541.1; R83000; AL161952.1; AK023843.1; AK021571.1; AK023783.1;AU147182; AL080112.1; AW971983; AI683552; NM_(—)024006.1; AK026565.1;NM_(—)014182.1; NM_(—)021800.1; NM_(—)016049.1; NM_(—)019023.1;NM_(—)021971.1; NM_(—)014128.1; AK025651.1; AA133341; and AF198444.1.

In another embodiment, the invention provides of a group of genes usefulin diagnosing lung diseases wherein the expression of the group of genesis higher in individuals exposed to air pollutants with cancer ascompared to individuals exposed to the same air pollutant who do nothave cancer, the group comprising probes that hybridize at least to 5,preferably at least about 5-10, still more preferably at least about10-20, still more preferably at least about 20-25, still more preferablyabout 25 genes consisting of transcripts (transcripts are identifiedusing their GenBank ID or Unigene ID numbers and the corresponding genenames appear in Table 1): NM_(—)000918; NM_(—)006430.1; NM_(—)001416.1;NM_(—)004090; NM_(—)006406.1; NM_(—)003001.2; NM_(—)006545.1;NM_(—)002437.1; NM_(—)006286; NM_(—)001123//NM_(—)006721; NM_(—)024824;NM_(—)004935.1; NM_(—)001696; NM_(—)005494///NM_(—)058246; NM_(—)006368;NM_(—)002268///NM_(—)032771; NM_(—)006694; NM_(—)004691; NM_(—)012394;NM_(—)021800; NM_(—)016049; NM_(—)138387; NM_(—)024531; andNM_(—)018509.

In another embodiment, the invention provides of a group of genes usefulin diagnosing lung diseases wherein the expression of the group of genesis higher in individuals exposed to air pollutants with cancer ascompared to individuals exposed to the same air pollutant who do nothave cancer, the group comprising probes that hybridize at least to 5,preferably at least about 5-10, still more preferably at least about10-20, still more preferably at least about 20-23, still more preferablyabout 23 genes consisting of transcripts (transcripts are identifiedusing their Genbank ID or Unigene ID numbers and the corresponding genenames appear in Table 2): NM_(—)014182.1; NM_(—)001281.1;NM_(—)024006.1; AF135421.1; L76200.1; NM_(—)000346.1; BC008710.1;BC000423.2; BC008710.1; NM_(—)007062; BC075839.1///BC073760.1;BC072436.1///BC004560.2; BC001016.2; BC005023.1; BC000360.2; BC007455.2;BC023528.2///BC047680.1; BC064957.1; BC008710.1; BC066329.1; BC023976.2;BC008591.2///BC050440.1///BC048096.1; and BC028912.1.

In another embodiment, the invention provides of a group of genes usefulin diagnosing lung diseases wherein the expression of the group of genesis higher in individuals exposed to air pollutants with cancer ascompared to individuals exposed to the same air pollutant who do nothave cancer, the group comprising probes that hybridize at least to 5,preferably at least about 5-10, still more preferably at least about10-20, still more preferably at least about 20-25, still more preferablyabout 25 genes consisting of transcripts (transcripts are identifiedusing their GenBank ID or Unigene ID numbers and the corresponding genenames appear in Table 3): NM_(—)007062.1; NM_(—)001281.1; BC000120.1;NM_(—)014255.1; BC002642.1; NM_(—)000346.1; NM_(—)006545.1; BG034328;NM_(—)021822.1; NM_(—)021069.1; NM_(—)019067.1; NM_(—)017925.1;NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1; AF126181.1; U93240.1;U90552.1; AF151056.1; U85430.1; U51007.1; BC005969.1; NM_(—)002271.1;AL566172; and AB014576.1.

In one embodiment, the invention provides a method of diagnosing lungdisease comprising the steps of measuring the expression profile of agene group in an individual suspected of being affected or being at highrisk of a lung disease (i.e. test individual), and comparing theexpression profile (i.e. control profile) to an expression profile of anindividual without the lung disease who has also been exposed to similarair pollutant than the test individual (i.e. control individual),wherein differences in the expression of genes when compared between theafore mentioned test individual and control individual of at least 10,more preferably at least 20, still more preferably at least 30, stillmore preferably at least 36, still more preferably between 36-180, stillmore preferably between 36-96, still more preferably between 36-84,still more preferably between 36-50, is indicative of the testindividual being affected with a lung disease. Groups of about 36 genesas shown in table 4, about 50 genes as shown in table 3, about 84 genesas shown in table 2 and about 96 genes as shown in table 1 arepreferred. The different gene groups can also be combined, so that thetest individual can be screened for all, three, two, or just one groupas shown in tables 1-4.

For example, if the expression profile of a test individual exposed tocigarette smoke is compared to the expression profile of the 50 genesshown in table 3, using the Affymetrix Inc. probe set on a gene chip asshown in table 3, the expression profile that is similar to the oneshown in FIG. 10 for the individuals with cancer, is indicative that thetest individual has cancer. Alternatively, if the expression profile ismore like the expression profile of the individuals who do not havecancer in FIG. 10, the test individual likely is not affected with lungcancer.

The group of 50 genes was identified using the GenePattern server fromthe Broad Institute, which includes the Weighted Voting algorithm. Thedefault settings, i.e., the signal to noise ratio and no gene filtering,were used. GenePattern is available through the World Wide Wed atlocation broad.mit.edu/cancer/software/genepattern. This program allowsanalysis of data in groups rather than as individual genes. Thus, in onepreferred embodiment, the expression of substantially all 50 genes ofTable 3, are analyzed together. The expression profile of lower thatnormal expression of genes selected from the group consisting ofBF218804; AK022494.1; AA114843; BE467941; NM_(—)003541.1; R83000;AL161952.1; AK023843.1; AK021571.1; AK023783.1; AU147182; AL080112.1;AW971983; A1683552; NM_(—)024006.1; AK026565.1; NM_(—)014182.1;NM_(—)021800.1; NM_(—)016049.1; NM_(—)019023.1; NM_(—)021971.1;NM_(—)014128.1; AK025651.1; AA133341; and AF198444.1, and the geneexpression profile of higher than normal expression of genes selectedfrom the group consisting of NM_(—)007062.1; NM_(—)001281.1; BC000120.1;NM_(—)014255.1; BC002642.1; NM_(—)000346.1; NM_(—)006545.1; BG034328;NM_(—)021822.1; NM_(—)021069.1; NM_(—)019067.1; NM_(—)017925.1;NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1; AF126181.1; U93240.1;U90552.1; AF151056.1; U85430.1; U51007.1; BC005969.1; NM_(—)002271.1;AL566172; and AB014576.1, is indicative of the individual having orbeing at high risk of developing lung disease, such as lung cancer. Inone preferred embodiment, the expression pattern of all the genes in theTable 3 is analyzed. In one embodiment, in addition to analyzing thegroup of predictor genes of Table 3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-15,15-20, 20-30, or more of the individual predictor genes identified usingthe t-test analysis are analyzed. Any combination of, for example, 5-10or more of the group predictor genes and 5-10, or more of the individualgenes can also be used.

The term “expression profile” as used herein, refers to the amount ofthe gene product of each of the analyzed individual genes in the sample.The “expression profile” is like a signature expression map, like theone shown for each individual in FIG. 10, on the Y-axis.

The term “lung disease”, as used herein, refers to disorders including,but not limited to, asthma, chronic bronchitis, emphysema,bronchietasis, primary pulmonary hypertension and acute respiratorydistress syndrome. The methods described herein may also be used todiagnose or treat lung disorders that involve the immune systemincluding, hypersensitivity pneumonitis, eosinophilic pneumonias, andpersistent fungal infections, pulmonary fibrosis, systemic sclerosis,idiopathic pulmonary hemosiderosis, pulmonary alveolar proteinosis,cancers of the lung such as adenocarcinoma, squamous cell carcinoma,small cell and large cell carcinomas, and benign neoplasm of the lungincluding bronchial adenomas and hamartomas. In one preferredembodiment, the lung disease is lung cancer.

The term “air pollutants”, as used herein, refers to any air impuritiesor environmental airway stress inducing agents, such as cigarette smoke,cigar smoke, smog, asbestos, and other air pollutants that havesuspected or proven association to lung diseases.

The term “individual”, as used herein, preferably refers to human.However, the methods are not limited to humans, and a skilled artisancan use the diagnostic/prognostic gene groupings of the presentinvention in, for example, laboratory test animals, preferably animalsthat have lungs, such as non-human primates, murine species, including,but not limited to rats and mice, dogs, sheep, pig, guinea pigs, andother model animals. Such laboratory tests can be used, for example inpre-clinical animal testing of drugs intended to be used to treat orprevent lung diseases.

The phrase “altered expression” as used herein, refers to eitherincreased or decreased expression in an individual exposed to airpollutant, such as a smoker, with cancer when compared to an expressionpattern of the lung cells from an individual exposed to similar airpollutant, such as smoker, who does not have cancer. Tables 1 and 2 showthe preferred expression pattern changes of the invention. The terms“up” and “down” in the tables refer to the amount of expression in asmoker with cancer to the amount of expression in a smoker withoutcancer. Similar expression pattern changes are likely associated withdevelopment of cancer in individuals who have been exposed to otherairway pollutants.

In one embodiment, the group of genes the expression of which isanalyzed in diagnosis and/or prognosis of lung cancer are selected fromthe group of 80 genes as shown in Table 5. Any combination of genes canbe selected from the 80 genes. In one embodiment, the combination of 20genes shown in Table 7 is selected. In one embodiment, a combination ofgenes from Table 6 is selected.

TABLE 5 Group of 80 genes for prognostic and diagnostic testing of lungcancer. Affymetrix Gene symbol Number of Signal to noise in a ID (HUGOID) runs* cancer sample** 200729_s_at ACTR2 736 −0.22284 200760_s_atARL6IP5 483 −0.21221 201399_s_at TRAM1 611 −0.21328 201444_s_at ATP6AP2527 −0.21487 201635_s_at FXR1 458 −0.2162 201689_s_at TPD52 565 −0.22292201925_s_at DAF 717 −0.25875 201926_s_at DAF 591 −0.23228 201946_s_atCCT2 954 −0.24592 202118_s_at CPNE3 334 −0.21273 202704_at TOB1 943−0.25724 202833_s_at SERPINA1 576 −0.20583 202935_s_at SOX9 750 −0.25574203413_at NELL2 629 −0.23576 203881_s_at DMD 850 −0.24341 203908_atSLC4A4 887 −0.23167 204006_s_at FCGR3A /// FCGR3B 207 −0.20071204403_x_at KIAA0738 923 0.167772 204427_s_at RNP24 725 −0.2366206056_x_at SPN 976 0.196398 206169_x_at RoXaN 984 0.259637 207730_x_atHDGF2 969 0.169108 207756_at — 855 0.161708 207791_s_at RAB1A 823−0.21704 207953_at AD7C-NTP 1000 0.218433 208137_x_at — 996 0.191938208246_x_at TK2 982 0.179058 208654_s_at CD164 388 −0.21228 208892_s_atDUSP6 878 −0.25023 209189_at FOS 935 −0.27446 209204_at LMO4 78 0.158674209267_s_at SLC39A8 228 −0.24231 209369_at ANXA3 384 −0.19972209656_s_at TMEM47 456 −0.23033 209774_x_at CXCL2 404 −0.2117 210145_atPLA2G4A 475 −0.26146 210168_at C6 458 −0.24157 210317_s_at YWHAE 803−0.29542 210397_at DEFB1 176 −0.22512 210679_x_at — 970 0.181718211506_s_at IL8 270 −0.3105 212006_at UBXD2 802 −0.22094 213089_atLOC153561 649 0.164097 213736_at COX5B 505 0.155243 213813_x_at — 7890.178643 214007_s_at PTK9 480 −0.21285 214146_s_at PPBP 593 −0.24265214594_x_at ATP8B1 962 0.284039 214707_x_at ALMS1 750 0.164047214715_x_at ZNF160 996 0.198532 215204_at SENP6 211 0.169986 215208_x_atRPL35A 999 0.228485 215385_at FTO 164 0.187634 215600_x_at FBXW12 9600.17329 215604_x_at UBE2D2 998 0.224878 215609_at STARD7 940 0.191953215628_x_at PPP2CA 829 0.16391 215800_at DUOX1 412 0.160036 215907_atBACH2 987 0.178338 215978_x_at LOC152719 645 0.163399 216834_at — 633−0.25508 216858_x_at — 997 0.232969 217446_x_at — 942 0.182612217653_x_at — 976 0.270552 217679_x_at — 987 0.265918 217715_x_atZNF354A 995 0.223881 217826_s_at UBE2J1 812 −0.23003 218155_x_atFLJ10534 998 0.186425 218976_at DNAJC12 486 −0.22866 219392_x_atFLJ11029 867 0.169113 219678_x_at DCLRE1C 877 0.169975 220199_s_atFLJ12806 378 −0.20713 220389_at FLJ23514 102 0.239341 220720_x_atFLJ14346 989 0.17976 221191_at DKFZP434A0131 616 0.185412 221310_atFGF14 511 −0.19965 221765_at — 319 −0.25025 222027_at NUCKS 547 0.171954222104_x_at GTF2H3 981 0.186025 222358_x_at — 564 0.194048

TABLE 6 Group of 535 genes useful in prognosis or diagnosis of lungcancer. Signal to Gene symbol Number of noise in a Affymetrix ID (HUGOID) runs* cancer sample** 200729_s_at ACTR2 736 −0.22284 200760_s_atARL6IP5 483 −0.21221 201399_s_at TRAM1 611 −0.21328 201444_s_at ATP6AP2527 −0.21487 201635_s_at FXR1 458 −0.2162 201689_s_at TPD52 565 −0.22292201925_s_at DAF 717 −0.25875 201926_s_at DAF 591 −0.23228 201946_s_atCCT2 954 −0.24592 202118_s_at CPNE3 334 −0.21273 202704_at TOB1 943−0.25724 202833_s_at SERPINA1 576 −0.20583 202935_s_at SOX9 750 −0.25574203413_at NELL2 629 −0.23576 203881_s_at DMD 850 −0.24341 203908_atSLC4A4 887 −0.23167 204006_s_at FCGR3A /// FCGR3B 207 −0.20071204403_x_at KIAA0738 923 0.167772 204427_s_at RNP24 725 −0.2366206056_x_at SPN 976 0.196398 206169_x_at RoXaN 984 0.259637 207730_x_atHDGF2 969 0.169108 207756_at — 855 0.161708 207791_s_at RAB1A 823−0.21704 207953_at AD7C-NTP 1000 0.218433 208137_x_at — 996 0.191938208246_x_at TK2 982 0.179058 208654_s_at CD164 388 −0.21228 208892_s_atDUSP6 878 −0.25023 209189_at FOS 935 −0.27446 209204_at LMO4 78 0.158674209267_s_at SLC39A8 228 −0.24231 209369_at ANXA3 384 −0.19972209656_s_at TMEM47 456 −0.23033 209774_x_at CXCL2 404 −0.2117 210145_atPLA2G4A 475 −0.26146 210168_at C6 458 −0.24157 210317_s_at YWHAE 803−0.29542 210397_at DEFB1 176 −0.22512 210679_x_at — 970 0.181718211506_s_at IL8 270 −0.3105 212006_at UBXD2 802 −0.22094 213089_atLOC153561 649 0.164097 213736_at COX5B 505 0.155243 213813_x_at — 7890.178643 214007_s_at PTK9 480 −0.21285 214146_s_at PPBP 593 −0.24265214594_x_at ATP8B1 962 0.284039 214707_x_at ALMS1 750 0.164047214715_x_at ZNF160 996 0.198532 215204_at SENP6 211 0.169986 215208_x_atRPL35A 999 0.228485 215385_at FTO 164 0.187634 215600_x_at FBXW12 9600.17329 215604_x_at UBE2D2 998 0.224878 215609_at STARD7 940 0.191953215628_x_at PPP2CA 829 0.16391 215800_at DUOX1 412 0.160036 215907_atBACH2 987 0.178338 215978_x_at LOC152719 645 0.163399 216834_at — 633−0.25508 216858_x_at — 997 0.232969 217446_x_at — 942 0.182612217653_x_at — 976 0.270552 217679_x_at — 987 0.265918 217715_x_atZNF354A 995 0.223881 217826_s_at UBE2J1 812 −0.23003 218155_x_atFLJ10534 998 0.186425 218976_at DNAJC12 486 −0.22866 219392_x_atFLJ11029 867 0.169113 219678_x_at DCLRE1C 877 0.169975 220199_s_atFLJ12806 378 −0.20713 220389_at FLJ23514 102 0.239341 220720_x_atFLJ14346 989 0.17976 221191_at DKFZP434A0131 616 0.185412 221310_atFGF14 511 −0.19965 221765_at — 319 −0.25025 222027_at NUCKS 547 0.171954222104_x_at GTF2H3 981 0.186025 222358_x_at — 564 0.194048 202113_s_atSNX2 841 −0.20503 207133_x_at ALPK1 781 0.155812 218989_x_at SLC30A5 765−0.198 200751_s_at HNRPC 759 −0.19243 220796_x_at SLC35E1 691 0.158199209362_at SURB7 690 −0.18777 216248_s_at NR4A2 678 −0.19796 203138_atHAT1 669 −0.18115 221428_s_at TBL1XR1 665 −0.19331 218172_s_at DERL1 665−0.16341 215861_at FLJ14031 651 0.156927 209288_s_at CDC42EP3 638−0.20146 214001_x_at RPS10 634 0.151006 209116_x_at HBB 626 −0.12237215595_x_at GCNT2 625 0.136319 208891_at DUSP6 617 −0.17282 215067_x_atPRDX2 616 0.160582 202918_s_at PREI3 614 −0.17003 211985_s_at CALM1 614−0.20103 212019_at RSL1D1 601 0.152717 216187_x_at KNS2 591 0.14297215066_at PTPRF 587 0.143323 212192_at KCTD12 581 −0.17535 217586_x_at —577 0.147487 203582_s_at RAB4A 567 −0.18289 220113_x_at POLR1B 5630.15764 217232_x_at HBB 561 −0.11398 201041_s_at DUSP1 560 −0.18661211450_s_at MSH6 544 −0.15597 202648_at RPS19 533 0.150087 202936_s_atSOX9 533 −0.17714 204426_at RNP24 526 −0.18959 206392_s_at RARRES1 517−0.18328 208750_s_at ARF1 515 −0.19797 202089_s_at SLC39A6 512 −0.19904211297_s_at CDK7 510 −0.15992 215373_x_at FLJ12151 509 0.146742213679_at FLJ13946 492 −0.10963 201694_s_at EGR1 490 −0.19478209142_s_at UBE2G1 487 −0.18055 217706_at LOC220074 483 0.11787212991_at FBXO9 476 0.148288 201289_at CYR61 465 −0.19925 206548_atFLJ23556 465 0.141583 202593_s_at MIR16 462 −0.17042 202932_at YES1 461−0.17637 220575_at FLJ11800 461 0.116435 217713_x_at DKFZP566N034 4520.145994 211953_s_at RANBP5 447 −0.17838 203827_at WIPI49 447 −0.17767221997_s_at MRPL52 444 0.132649 217662_x_at BCAP29 434 0.116886218519_at SLC35A5 428 −0.15495 214833_at KIAA0792 428 0.132943201339_s_at SCP2 426 −0.18605 203799_at CD302 422 −0.16798 211090_s_atPRPF4B 421 −0.1838 220071_x_at C15orf25 420 0.138308 203946_s_at ARG2415 −0.14964 213544_at ING1L 415 0.137052 209908_s_at — 414 0.131346201688_s_at TPD52 410 −0.18965 215587_x_at BTBD14B 410 0.139952201699_at PSMC6 409 −0.13784 214902_x_at FLJ42393 409 0.140198214041_x_at RPL37A 402 0.106746 203987_at FZD6 392 −0.19252 211696_x_atHBB 392 −0.09508 218025_s_at PECI 389 −0.18002 215852_x_at KIAA0889 3820.12243 209458_x_at HBA1 /// HBA2 380 −0.09796 219410_at TMEM45A 379−0.22387 215375_x_at — 379 0.148377 206302_s_at NUDT4 376 −0.18873208783_s_at MCP 372 −0.15076 211374_x_at — 364 0.131101 220352_x_atMGC4278 364 0.152722 216609_at TXN 363 0.15162 201942_s_at CPD 363−0.1889 202672_s_at ATF3 361 −0.12935 204959_at MNDA 359 −0.21676211996_s_at KIAA0220 358 0.144358 222035_s_at PAPOLA 353 −0.14487208808_s_at HMGB2 349 −0.15222 203711_s_at HIBCH 347 −0.13214215179_x_at PGF 347 0.146279 213562_s_at SQLE 345 −0.14669 203765_at GCA340 −0.1798 214414_x_at HBA2 336 −0.08492 217497_at ECGF1 336 0.123255220924_s_at SLC38A2 333 −0.17315 218139_s_at C14orf108 332 −0.15021201096_s_at ARF4 330 −0.18887 220361_at FLJ12476 325 −0.15452202169_s_at AASDHPPT 323 −0.15787 202527_s_at SMAD4 322 −0.18399202166_s_at PPP1R2 320 −0.16402 204634_at NEK4 319 −0.15511 215504_x_at— 319 0.145981 202388_at RGS2 315 −0.14894 215553_x_at WDR45 3150.137586 200598_s_at TRA1 314 −0.19349 202435_s_at CYP1B1 313 0.056937216206_x_at MAP2K7 313 0.10383 212582_at OSBPL8 313 −0.17843 216509_x_atMLLT10 312 0.123961 200908_s_at RPLP2 308 0.136645 215108_x_at TNRC9 306−0.1439 213872_at C6orf62 302 −0.19548 214395_x_at EEF1D 302 0.128234222156_x_at CCPG1 301 −0.14725 201426_s_at VIM 301 −0.17461 221972_s_atCab45 299 −0.1511 219957_at — 298 0.130796 215123_at — 295 0.125434212515_s_at DDX3X 295 −0.14634 203357_s_at CAPN7 295 −0.17109211711_s_at PTEN 295 −0.12636 206165_s_at CLCA2 293 −0.17699 213959_s_atKIAA1005 289 −0.16592 215083_at PSPC1 289 0.147348 219630_at PDZK1IP1287 −0.15086 204018_x_at HBA1 /// HBA2 286 −0.08689 208671_at TDE2 286−0.17839 203427_at ASF1A 286 −0.14737 215281_x_at POGZ 286 0.142825205749_at CYP1A1 285 0.107118 212585_at OSBPL8 282 −0.13924 211745_x_atHBA1 /// HBA2 281 −0.08437 208078_s_at SNF1LK 278 −0.14395 218041_x_atSLC38A2 276 −0.17003 212588_at PTPRC 270 −0.1725 212397_at RDX 270−0.15613 208268_at ADAM28 269 0.114996 207194_s_at ICAM4 269 0.127304222252_x_at — 269 0.132241 217414_x_at HBA2 266 −0.08974 207078_at MED6261 0.1232 215268_at KIAA0754 261 0.13669 221387_at GPR147 261 0.128737201337_s_at VAMP3 259 −0.17284 220218_at C9orf68 259 0.125851 222356_atTBL1Y 259 0.126765 208579_x_at H2BFS 258 −0.16608 219161_s_at CKLF 257−0.12288 202917_s_at S100A8 256 −0.19869 204455_at DST 255 −0.13072211672_s_at ARPC4 254 −0.17791 201132_at HNRPH2 254 −0.12817 218313_s_atGALNT7 253 −0.179 218930_s_at FLJ11273 251 −0.15878 219166_at C14orf104250 −0.14237 212805_at KIAA0367 248 −0.16649 201551_s_at LAMP1 247−0.18035 202599_s_at NRIP1 247 −0.16226 203403_s_at RNF6 247 −0.14976214261_s_at ADH6 242 −0.1414 202033_s_at RB1CC1 240 −0.18105 203896_s_atPLCB4 237 −0.20318 209703_x_at DKFZP586A0522 234 0.140153 211699_x_atHBA1 /// HBA2 232 −0.08369 210764_s_at CYR61 231 −0.13139 206391_atRARRES1 230 −0.16931 201312_s_at SH3BGRL 225 −0.12265 200798_x_at MCL1221 −0.13113 214912_at — 221 0.116262 204621_s_at NR4A2 217 −0.10896217761_at MTCBP-1 217 −0.17558 205830_at CLGN 216 −0.14737 218438_s_atMED28 214 −0.14649 207475_at FABP2 214 0.097003 208621_s_at VIL2 213−0.19678 202436_s_at CYP1B1 212 0.042216 202539_s_at HMGCR 210 −0.15429210830_s_at PON2 209 −0.17184 211906_s_at SERPINB4 207 −0.14728202241_at TRIB1 207 −0.10706 203594_at RTCD1 207 −0.13823 215863_at TFR2207 0.095157 221992_at LOC283970 206 0.126744 221872_at RARRES1 205−0.11496 219564_at KCNJ16 205 −0.13908 201329_s_at ETS2 205 −0.14994214188_at HIS1 203 0.1257 201667_at GJA1 199 −0.13848 201464_x_at JUN199 −0.09858 215409_at LOC254531 197 0.094182 202583_s_at RANBP9 197−0.13902 215594_at — 197 0.101007 214326_x_at JUND 196 −0.1702217140_s_at VDAC1 196 −0.14682 215599_at SMA4 195 0.133438 209896_s_atPTPN11 195 −0.16258 204846_at CP 195 −0.14378 222303_at — 193 −0.10841218218_at DIP13B 193 −0.12136 211015_s_at HSPA4 192 −0.13489 208666_s_atST13 191 −0.13361 203191_at ABCB6 190 0.096808 202731_at PDCD4 190−0.1545 209027_s_at ABI1 190 −0.15472 205979_at SCGB2A1 189 −0.15091216351_x_at DAZ1 /// DAZ3 /// 189 0.106368 DAZ2 /// DAZ4 220240_s_atC13orf11 188 −0.16959 204482_at CLDN5 187 0.094134 217234_s_at VIL2 186−0.16035 214350_at SNTB2 186 0.095723 201693_s_at EGR1 184 −0.10732212328_at KIAA1102 182 −0.12113 220168_at CASC1 181 −0.1105 203628_atIGF1R 180 0.067575 204622_x_at NR4A2 180 −0.11482 213246_at C14orf109180 −0.16143 218728_s_at HSPC163 180 −0.13248 214753_at PFAAP5 1790.130184 206336_at CXCL6 178 −0.05634 201445_at CNN3 178 −0.12375209886_s_at SMAD6 176 0.079296 213376_at ZBTB1 176 −0.17777 213887_s_atPOLR2E 175 −0.16392 204783_at MLF1 174 −0.13409 218824_at FLJ10781 1730.1394 212417_at SCAMP1 173 −0.17052 202437_s_at CYP1B1 171 0.033438217528_at CLCA2 169 −0.14179 218170_at ISOC1 169 −0.14064 206278_atPTAFR 167 0.087096 201939_at PLK2 167 −0.11049 200907_s_at KIAA0992 166−0.18323 207480_s_at MEIS2 166 −0.15232 201417_at SOX4 162 −0.09617213826_s_at — 160 0.097313 214953_s_at APP 159 −0.1645 204897_at PTGER4159 −0.08152 201711_x_at RANBP2 158 −0.17192 202457_s_at PPP3CA 158−0.18821 206683_at ZNF165 158 −0.08848 214581_x_at TNFRSF21 156 −0.14624203392_s_at CTBP1 155 −0.16161 212720_at PAPOLA 155 −0.14809 207758_atPPM1F 155 0.090007 220995_at STXBP6 155 0.106749 213831_at HLA-DQA1 1540.193368 212044_s_at — 153 0.098889 202434_s_at CYP1B1 153 0.049744206166_s_at CLCA2 153 −0.1343 218343_s_at GTF3C3 153 −0.13066 202557_atSTCH 152 −0.14894 201133_s_at PJA2 152 −0.18481 213605_s_at MGC22265 1510.130895 210947_s_at MSH3 151 −0.12595 208310_s_at C7orf28A /// C7orf28B151 −0.15523 209307_at — 150 −0.1667 215387_x_at GPC6 148 0.114691213705_at MAT2A 147 0.104855 213979_s_at — 146 0.121562 212731_atLOC157567 146 −0.1214 210117_at SPAG1 146 −0.11236 200641_s_at YWHAZ 145−0.14071 210701_at CFDP1 145 0.151664 217152_at NCOR1 145 0.130891204224_s_at GCH1 144 −0.14574 202028_s_at — 144 0.094276 201735_s_atCLCN3 144 −0.1434 208447_s_at PRPS1 143 −0.14933 220926_s_at C1orf22 142−0.17477 211505_s_at STAU 142 −0.11618 221684_s_at NYX 142 0.102298206906_at ICAM5 141 0.076813 213228_at PDE8B 140 −0.13728 217202_s_atGLUL 139 −0.15489 211713_x_at KIAA0101 138 0.108672 215012_at ZNF451 1380.13269 200806_s_at HSPD1 137 −0.14811 201466_s_at JUN 135 −0.0667211564_s_at PDLIM4 134 −0.12756 207850_at CXCL3 133 −0.17973 221841_s_atKLF4 133 −0.1415 200605_s_at PRKAR1A 132 −0.15642 221198_at SCT 1320.08221 201772_at AZIN1 131 −0.16639 205009_at TFF1 130 −0.17578205542_at STEAP1 129 −0.08498 218195_at C6orf211 129 −0.14497 213642_at— 128 0.079657 212891_s_at GADD45GIP1 128 −0.09272 202798_at SEC24B 127−0.12621 222207_x_at — 127 0.10783 202638_s_at ICAM1 126 0.070364200730_s_at PTP4A1 126 −0.15289 219355_at FLJ10178 126 −0.13407220266_s_at KLF4 126 −0.15324 201259_s_at SYPL 124 −0.16643 209649_atSTAM2 124 −0.1696 220094_s_at C6orf79 123 −0.12214 221751_at PANK3 123−0.1723 200008_s_at GDI2 123 −0.15852 205078_at PIGF 121 −0.13747218842_at FLJ21908 121 −0.08903 202536_at CHMP2B 121 −0.14745 220184_atNANOG 119 0.098142 201117_s_at CPE 118 −0.20025 219787_s_at ECT2 117−0.14278 206628_at SLC5A1 117 −0.12838 204007_at FCGR3B 116 −0.15337209446_s_at — 116 0.100508 211612_s_at IL13RA1 115 −0.17266 220992_s_atC1orf25 115 −0.11026 221899_at PFAAP5 115 0.11698 221719_s_at LZTS1 1150.093494 201473_at JUNB 114 −0.10249 221193_s_at ZCCHC10 112 −0.08003215659_at GSDML 112 0.118288 205157_s_at KRT17 111 −0.14232 201001_s_atUBE2V1 /// Kua-UEV 111 −0.16786 216789_at — 111 0.105386 205506_at VIL1111 0.097452 204875_s_at GMDS 110 −0.12995 207191_s_at ISLR 110 0.100627202779_s_at UBE2S 109 −0.11364 210370_s_at LY9 109 0.096323 202842_s_atDNAJB9 108 −0.15326 201082_s_at DCTN1 107 −0.10104 215588_x_at RIOK3 1070.135837 211076_x_at DRPLA 107 0.102743 210230_at — 106 0.115001206544_x_at SMARCA2 106 −0.12099 208852_s_at CANX 105 −0.14776 215405_atMYO1E 105 0.086393 208653_s_at CD164 104 −0.09185 206355_at GNAL 1030.1027 210793_s_at NUP98 103 −0.13244 215070_x_at RABGAP1 103 0.125029203007_x_at LYPLA1 102 −0.17961 203841_x_at MAPRE3 102 −0.13389206759_at FCER2 102 0.081733 202232_s_at GA17 102 −0.11373 215892_at —102 0.13866 214359_s_at HSPCB 101 −0.12276 215810_x_at DST 101 0.098963208937_s_at ID1 100 −0.06552 213664_at SLC1A1 100 −0.12654 219338_s_atFLJ20156 100 −0.10332 206595_at CST6 99 −0.10059 207300_s_at F7 990.082445 213792_s_at INSR 98 0.137962 209674_at CRY1 98 −0.1381840665_at FMO3 97 −0.05976 217975_at WBP5 97 −0.12698 210296_s_at PXMP397 −0.13537 215483_at AKAP9 95 0.125966 212633_at KIAA0776 95 −0.16778206164_at CLCA2 94 −0.13117 216813_at — 94 0.089023 208925_at C3orf4 94−0.1721 219469_at DNCH2 94 −0.12003 206016_at CXorf37 93 −0.11569216745_x_at LRCH1 93 0.117149 212999_x_at HLA-DQB1 92 0.110258216859_x_at — 92 0.116351 201636_at — 92 −0.13501 204272_at LGALS4 920.110391 215454_x_at SFTPC 91 0.064918 215972_at — 91 0.097654220593_s_at FLJ20753 91 0.095702 222009_at CGI-14 91 0.070949207115_x_at MBTD1 91 0.107883 216922_x_at DAZ1 /// DAZ3 /// 91 0.086888DAZ2 /// DAZ4 217626_at AKR1C1 /// AKR1C2 90 0.036545 211429_s_atSERPINA1 90 −0.11406 209662_at CETN3 90 −0.10879 201629_s_at ACP1 90−0.14441 201236_s_at BTG2 89 −0.09435 217137_x_at — 89 0.070954212476_at CENTB2 89 −0.1077 218545_at FLJ11088 89 −0.12452 208857_s_atPCMT1 89 −0.14704 221931_s_at SEH1L 88 −0.11491 215046_at FLJ23861 88−0.14667 220222_at PRO1905 88 0.081524 209737_at AIP1 87 −0.07696203949_at MPO 87 0.113273 219290_x_at DAPP1 87 0.111366 205116_at LAMA286 0.05845 222316_at VDP 86 0.091505 203574_at NFIL3 86 −0.14335207820_at ADH1A 86 0.104444 203751_x_at JUND 85 −0.14118 202930_s_atSUCLA2 85 −0.14884 215404_x_at FGFR1 85 0.119684 216266_s_at ARFGEF1 85−0.12432 212806_at KIAA0367 85 −0.13259 219253_at — 83 −0.14094214605_x_at GPR1 83 0.114443 205403_at IL1R2 82 −0.19721 222282_at PAPD482 0.128004 214129_at PDE4DIP 82 −0.13913 209259_s_at CSPG6 82 −0.12618216900_s_at CHRNA4 82 0.105518 221943_x_at RPL38 80 0.086719 215386_atAUTS2 80 0.129921 201990_s_at CREBL2 80 −0.13645 220145_at FLJ21159 79−0.16097 221173_at USH1C 79 0.109348 214900_at ZKSCAN1 79 0.075517203290_at HLA-DQA1 78 −0.20756 215382_x_at TPSAB1 78 −0.09041201631_s_at IER3 78 −0.12038 212188_at KCTD12 77 −0.14672 220428_atCD207 77 0.101238 215349_at — 77 0.10172 213928_s_at HRB 77 0.092136221228_s_at — 77 0.0859 202069_s_at IDH3A 76 −0.14747 208554_at POU4F376 0.107529 209504_s_at PLEKHB1 76 −0.13125 212989_at TMEM23 75 −0.11012216197_at ATF7IP 75 0.115016 204748_at PTGS2 74 −0.15194 205221_at HGD74 0.096171 214705_at INADL 74 0.102919 213939_s_at RIPX 74 0.091175203691_at PI3 73 −0.14375 220532_s_at LR8 73 −0.11682 209829_at C6orf3273 −0.08982 206515_at CYP4F3 72 0.104171 218541_s_at C8orf4 72 −0.09551210732_s_at LGALS8 72 −0.13683 202643_s_at TNFAIP3 72 −0.16699218963_s_at KRT23 72 −0.10915 213304_at KIAA0423 72 −0.12256 202768_atFOSB 71 −0.06289 205623_at ALDH3A1 71 0.045457 206488_s_at CD36 71−0.15899 204319_s_at RGS10 71 −0.10107 217811_at SELT 71 −0.16162202746_at ITM2A 70 −0.06424 221127_s_at RIG 70 0.110593 209821_atC9orf26 70 −0.07383 220957_at CTAGE1 70 0.092986 215577_at UBE2E1 700.10305 214731_at DKFZp547A023 70 0.102821 210512_s_at VEGF 69 −0.11804205267_at POU2AF1 69 0.101353 216202_s_at SPTLC2 69 −0.11908 220477_s_atC20orf30 69 −0.16221 205863_at S100A12 68 −0.10353 215780_s_at SET ///LOC389168 68 −0.10381 218197_s_at OXR1 68 −0.14424 203077_s_at SMAD2 68−0.11242 222339_x_at — 68 0.121585 200698_at KDELR2 68 −0.15907210540_s_at B4GALT4 67 −0.13556 217725_x_at PAI-RBP1 67 −0.14956217082_at — 67 0.086098

TABLE 7 Group of 20 genes useful in prognosis and/or diagnosis of lungcancer. Gene symbol Signal to noise Affymetrix ID HUGO ID Number ofruns* in a cancer sample* 207953_at AD7C-NTP 1000 0.218433 215208_x_atRPL35A 999 0.228485 215604_x_at UBE2D2 998 0.224878 218155_x_at FLJ10534998 0.186425 216858_x_at — 997 0.232969 208137_x_at — 996 0.191938214715_x_at ZNF160 996 0.198532 217715_x_at ZNF354A 995 0.223881220720_x_at FLJ14346 989 0.17976 215907_at BACH2 987 0.178338217679_x_at — 987 0.265918 206169_x_at RoXaN 984 0.259637 208246_x_atTK2 982 0.179058 222104_x_at GTF2H3 981 0.186025 206056_x_at SPN 9760.196398 217653_x_at — 976 0.270552 210679_x_at — 970 0.181718207730_x_at HDGF2 969 0.169108 214594_x_at ATP8B1 962 0.284039 *Thenumber of runs when the gene is indicated in cancer samples asdifferentially expressed out of 1000 test runs. ** Negative valuesindicate increase of expression in lung cancer, positive values indicatedecrease of expression in lung cancer.

One can use the above tables to correlate or compare the expression ofthe transcript to the expression of the gene product, i.e. protein.Increased expression of the transcript as shown in the table correspondsto increased expression of the gene product. Similarly, decreasedexpression of the transcript as shown in the table corresponds todecreased expression of the gene product.

In one preferred embodiment, one uses at least one, preferably at least2, 3, 4, 5, 6, 7, 8, 9, 10 or more, of the genes as listed in Tables 8,9 and/or 10. In one embodiment, one uses maximum of 500, 400, 300, 200,100, or 50 of the gene that include at least 5, 6, 7, 8, 9, 10-20,20-30, 30-40, 40-50, 50-60, 60-70, 1-70, of the genes listed in Tables8-10.

TABLE 8 361 Airway t-test gene list AffyID GeneName (HUGO ID)202437_s_at CYP1B1 206561_s_at AKR1B10 202436_s_at CYP1B1 205749_atCYP1A1 202435_s_at CYP1B1 201884_at CEACAM5 205623_at ALDH3A1 217626_at— 209921_at SLC7A11 209699_x_at AKR1C2 201467_s_at NQO1 201468_s_at NQO1202831_at GPX2 214303_x_at MUC5AC 211653_x_at AKR1C2 214385_s_at MUC5AC216594_x_at AKR1C1 205328_at CLDN10 209160_at AKR1C3 210519_s_at NQO1217678_at SLC7A11 205221_at HGD /// LOC642252 204151_x_at AKR1C1207469_s_at PIR 206153_at CYP4F11 205513_at TCN1 209386_at TM4SF1209351_at KRT14 204059_s_at ME1 209213_at CBR1 210505_at ADH7214404_x_at SPDEF 204058_at ME1 218002_s_at CXCL14 205499_at SRPX2210065_s_at UPK1B 204341_at TRIM16 /// TRIM16L /// LOC653524 221841_s_atKLF4 208864_s_at TXN 208699_x_at TKT 210397_at DEFB1 204971_at CSTA211657_at CEACAM6 201463_s_at TALDO1 214164_x_at CA12 203925_at GCLM201118_at PGD 201266_at TXNRD1 203757_s_at CEACAM6 202923_s_at GCLC214858_at GPC1 205009_at TFF1 219928_s_at CABYR 203963_at CA12210064_s_at UPK1B 219956_at GALNT6 208700_s_at TKT 203824_at TSPAN8207126_x_at UGT1A10 /// UGT1A8 /// UGT1A7 /// UGT1A6 /// UGT1A213441_x_at SPDEF 207430_s_at MSMB 209369_at ANXA3 217187_at MUC5AC209101_at CTGF 212221_x_at IDS 215867_x_at CA12 214211_at FTH1 217755_atHN1 201431_s_at DPYSL3 204875_s_at GMDS 215125_s_at UGT1A10 /// UGT1A8/// UGT1A7 /// UGT1A6 /// UGT1A 63825_at ABHD2 202922_at GCLC218313_s_at GALNT7 210297_s_at MSMB 209448_at HTATIP2 204532_x_atUGT1A10 /// UGT1A8 /// UGT1A7 /// UGT1A6 /// UGT1A 200872_at S100A10216351_x_at DAZ1 /// DAZ3 /// DAZ2 /// DAZ4 212223_at IDS 208680_atPRDX1 206515_at CYP4F3 208596_s_at UGT1A10 /// UGT1A8 /// UGT1A7 ///UGT1A6 /// UGT1A 209173_at AGR2 204351_at S100P 202785_at NDUFA7204970_s_at MAFG 222016_s_at ZNF323 200615_s_at AP2B1 206094_x_at UGT1A6209706_at NKX3-1 217977_at SEPX1 201487_at CTSC 219508_at GCNT3204237_at GULP1 213455_at LOC283677 213624_at SMPDL3A 206770_s_atSLC35A3 217975_at WBP5 201263_at TARS 218696_at EIF2AK3 212560_atC11orf32 218885_s_at GALNT12 212326_at VPS13D 217955_at BCL2L13203126_at IMPA2 214106_s_at GMDS 209309_at AZGP1 205112_at PLCE1215363_x_at FOLH1 206302_s_at NUDT4 /// NUDT4P1 200916_at TAGLN2205042_at GNE 217979_at TSPAN13 203397_s_at GALNT3 209786_at HMGN4211733_x_at SCP2 207222_at PLA2G10 204235_s_at GULP1 205726_at DIAPH2203911_at RAP1GAP 200748_s_at FTH1 212449_s_at LYPLA1 213059_at CREB3L1201272_at AKR1B1 208731_at RAB2 205979_at SCGB2A1 212805_at KIAA0367202804_at ABCC1 218095_s_at TPARL 205566_at ABHD2 209114_at TSPAN1202481_at DHRS3 202805_s_at ABCC1 219117_s_at FKBP11 213172_at TTC9202554_s_at GSTM3 218677_at S100A14 203306_s_at SLC35A1 204076_at ENTPD4200654_at P4HB 204500_s_at AGTPBP1 208918_s_at NADK 221485_at B4GALT5221511_x_at CCPG1 200733_s_at PTP4A1 217901_at DSG2 202769_at CCNG2202119_s_at CPNE3 200945_s_at SEC31L1 200924_s_at SLC3A2 208736_at ARPC3221556_at CDC14B 221041_s_at SLC17A5 215071_s_at HIST1H2AC 209682_atCBLB 209806_at HIST1H2BK 204485_s_at TOM1L1 201666_at TIMP1 203192_atABCB6 202722_s_at GFPT1 213135_at TIAM1 203509_at SORL1 214620_x_at PAM208919_s_at NADK 212724_at RND3 212160_at XPOT 212812_at SERINC5200696_s_at GSN 217845_x_at HIGD1A 208612_at PDIA3 219288_at C3orf14201923_at PRDX4 211960_s_at RAB7 64942_at GPR153 201659_s_at ARL1202439_s_at IDS 209249_s_at GHITM 218723_s_at RGC32 200087_s_at TMED2209694_at PTS 202320_at GTF3C1 201193_at IDH1 212233_at — 213891_s_at —203041_s_at LAMP2 202666_s_at ACTL6A 200863_s_at RAB11A 203663_s_atCOX5A 211404_s_at APLP2 201745_at PTK9 217823_s_at UBE2J1 202286_s_atTACSTD2 212296_at PSMD14 211048_s_at PDIA4 214429_at MTMR6 219429_atFA2H 212181_s_at NUDT4 222116_s_at TBC1D16 221689_s_at PIGP 209479_atCCDC28A 218434_s_at AACS 214665_s_at CHP 202085_at TJP2 217992_s_atEFHD2 203162_s_at KATNB1 205406_s_at SPA17 203476_at TPBG 201724_s_atGALNT1 200599_s_at HSP90B1 200929_at TMED10 200642_at SOD1 208946_s_atBECN1 202562_s_at C14orf1 201098_at COPB2 221253_s_at TXNDC5 201004_atSSR4 203221_at TLE1 201588_at TXNL1 218684_at LRRC8D 208799_at PSMB5201471_s_at SQSTM1 204034_at ETHE1 208689_s_at RPN2 212665_at TIPARP200625_s_at CAP1 213220_at LOC92482 200709_at FKBP1A 203279_at EDEM1200068_s_at CANX 200620_at TMEM59 200075_s_at GUK1 209679_s_at LOC57228210715_s_at SPINT2 209020_at C20orf111 208091_s_at ECOP 200048_s_at JTB218194_at REXO2 209103_s_at UFD1L 208718_at DDX17 219241_x_at SSH3216210_x_at TRIOBP 50277_at GGA1 218023_s_at FAM53C 32540_at PPP3CC43511_s_at — 212001_at SFRS14 208637_x_at ACTN1 201997_s_at SPEN205073_at CYP2J2 40837_at TLE2 204447_at ProSAPiP1 204604_at PFTK1210273_at PCDH7 208614_s_at FLNB 206510_at SIX2 200675_at CD81 219228_atZNF331 209426_s_at AMACR 204000_at GNB5 221742_at CUGBP1 208883_at EDD1210166_at TLR5 211026_s_at MGLL 220446_s_at CHST4 207636_at SERPINI2212226_s_at PPAP2B 210347_s_at BCL11A 218424_s_at STEAP3 204287_atSYNGR1 205489_at CRYM 36129_at RUTBC1 215418_at PARVA 213029_at NFIB221016_s_at TCF7L1 209737_at MAGI2 220389_at CCDC81 213622_at COL9A2204740_at CNKSR1 212126_at — 207760_s_at NCOR2 205258_at INHBB 213169_at— 33760_at PEX14 220968_s_at TSPAN9 221792_at RAB6B 205752_s_at GSTM5218974_at FLJ10159 221748_s_at TNS1 212185_x_at MT2A 209500_x_at TNFSF13/// TNFSF12-TNFSF13 215445_x_at 1-Mar 220625_s_at ELF5 32137_at JAG2219747_at FLJ23191 201397_at PHGDH 207913_at CYP2F1 217853_at TNS31598_g_at GAS6 203799_at CD302 203329_at PTPRM 208712_at CCND1210314_x_at TNFSF13 /// TNFSF12-TNFSF13 213217_at ADCY2 200953_s_atCCND2 204326_x_at MT1X 213488_at SNED1 213505_s_at SFRS14 200982_s_atANXA6 211732_x_at HNMT 202587_s_at AK1 396_f_at EPOR 200878_at EPAS1213228_at PDE8B 215785_s_at CYFIP2 213601_at SLIT1 37953_s_at ACCN2205206_at KAL1 212859_x_at MT1E 217165_x_at MT1F 204754_at HLF 218225_atSITPEC 209784_s_at JAG2 211538_s_at HSPA2 211456_x_at LOC650610204734_at KRT15 201563_at SORD 202746_at ITM2A 218025_s_at PECI203914_x_at HPGD 200884_at CKB 204753_s_at HLF 207718_x_at CYP2A6 ///CYP2A7 /// CYP2A7P1 /// CYP2A13 218820_at C14orf132 204745_x_at MT1G204379_s_at FGFR3 207808_s_at PROS1 207547_s_at FAM107A 208581_x_at MT1X205384_at FXYD1 213629_x_at MT1F 823_at CX3CL1 203687_at CX3CL1211295_x_at CYP2A6 204755_x_at HLF 209897_s_at SLIT2 40093_at BCAM211726_s_at FMO2 206461_x_at MT1H 219250_s_at FLRT3 210524_x_at —220798_x_at PRG2 219410_at TMEM45A 205680_at MMP10 217767_at C3 ///LOC653879 220562_at CYP2W1 210445_at FABP6 205725_at SCGB1A1 213432_atMUC5B /// LOC649768 209074_s_at FAM107A 216346_at SEC14L3

TABLE 9 107 Nose Leading Edge Genes AffxID Hugo ID 203369_x_at —218434_s_at AACS 205566_at ABHD2 217687_at ADCY2 210505_at ADH7205623_at ALDH3A1 200615_s_at AP2B1 214875_x_at APLP2 212724_at ARHE201659_s_at ARL1 208736_at ARPC3 213624_at ASM3A 209309_at AZGP1217188_s_at C14orf1 200620_at C1orf8 200068_s_at CANX 213798_s_at CAP1200951_s_at CCND2 202769_at CCNG2 201884_at CEACAM5 203757_s_at CEACAM6214665_s_at CHP 205328_at CLDN10 203663_s_at COX5A 202119_s_at CPNE3221156_x_at CPR8 201487_at CTSC 205749_at CYP1A1 207913_at CYP2F1206153_at CYP4F11 206514_s_at CYP4F3 216351_x_at DAZ4 203799_at DCL-1212665_at DKFZP434J214 201430_s_at DPYSL3 211048_s_at ERP70 219118_atFKBP11 214119_s_at FKBP1A 208918_s_at FLJ13052 217487_x_at FOLH1200748_s_at FTH1 201723_s_at GALNT1 218885_s_at GALNT12 203397_s_atGALNT3 218313_s_at GALNT7 203925_at GCLM 219508_at GCNT3 202722_s_atGFPT1 204875_s_at GMDS 205042_at GNE 208612_at GRP58 214040_s_at GSN214307_at HGD 209806_at HIST1H2BK 202579_x_at HMGN4 207180_s_at HTATIP2206342_x_at IDS 203126_at IMPA2 210927_x_at JTB 203163_at KATNB1204017_at KDELR3 213174_at KIAA0227 212806_at KIAA0367 210616_s_atKIAA0905 221841_s_at KLF4 203041_s_at LAMP2 213455_at LOC92689 218684_atLRRC5 204059_s_at ME1 207430_s_at MSMB 210472_at MT1G 213432_at MUC5B211498_s_at NKX3-1 201467_s_at NQO1 206303_s_at NUDT4 213498_at OASIS200656_s_at P4HB 213441_x_at PDEF 207469_s_at PIR 207222_at PLA2G10209697_at PPP3CC 201923_at PRDX4 200863_s_at RAB11A 208734_x_at RAB2203911_at RAP1GA1 218723_s_at RGC32 200087_s_at RNP24 200872_at S100A10205979_at SCGB2A1 202481_at SDR1 217977_at SEPX1 221041_s_at SLC17A5203306_s_at SLC35A1 207528_s_at SLC7A11 202287_s_at TACSTD2 210978_s_atTAGLN2 205513_at TCN1 201666_at TIMP1 208699_x_at TKT 217979_at TM4SF13203824_at TM4SF3 200929_at TMP21 221253_s_at TXNDC5 217825_s_at UBE2J1215125_s_at UGT1A10 210064_s_at UPK1B 202437_s_at CYP1B1

TABLE 10 70 gene list AFFYID Gene Name (HUGO ID) 213693_s_at MUC1211695_x_at MUC1 207847_s_at MUC1 208405_s_at CD164 220196_at MUC16217109_at MUC4 217110_s_at MUC4 204895_x_at MUC4 214385_s_at MUC5AC1494_f_at CYP2A6 210272_at CYP2B7P1 206754_s_at CYP2B7P1 210096_atCYP4B1 208928_at POR 207913_at CYP2F1 220636_at DNAI2 201999_s_at DYNLT1205186_at DNALI1 220125_at DNAI1 210345_s_at DNAH9 214222_at DNAH7211684_s_at DYNC1I2 211928_at DYNC1H1 200703_at DYNLL1 217918_at DYNLRB1217917_s_at DYNLRB1 209009_at ESD 204418_x_at GSTM2 215333_x_at GSTM1217751_at GSTK1 203924_at GSTA1 201106_at GPX4 200736_s_at GPX1204168_at MGST2 200824_at GSTP1 211630_s_at GSS 201470_at GSTO1201650_at KRT19 209016_s_at KRT7 209008_x_at KRT8 201596_x_at KRT18210633_x_at KRT10 207023_x_at KRT10 212236_x_at KRT17 201820_at KRT5204734_at KRT15 203151_at MAP1A 200713_s_at MAPRE1 204398_s_at EML240016_g_at MAST4 208634_s_at MACF1 205623_at ALDH3A1 212224_at ALDH1A1205640_at ALDH3B1 211004_s_at ALDH3B1 202054_s_at ALDH3A2 205208_atALDH1L1 201612_at ALDH9A1 201425_at ALDH2 201090_x_at K-ALPHA-1202154_x_at TUBB3 202477_s_at TUBGCP2 203667_at TBCA 204141_at TUBB2A207490_at TUBA4 208977_x_at TUBB2C 209118_s_at TUBA3 209251_x_at TUBA6211058_x_at K-ALPHA-1 211072_x_at K-ALPHA-1 211714_x_at TUBB 211750_x_atTUBA6 212242_at TUBA1 212320_at TUBB 212639_x_at K-ALPHA-1 213266_at 76P213476_x_at TUBB3 213646_x_at K-ALPHA-1 213726_x_at TUBB2C

Additionally, one can use any one or a combination of the genes listedin Table 9.

The analysis of the gene expression of one or more genes and/ortranscripts of the groups or their subgroups of the present inventioncan be performed using any gene expression method known to one skilledin the art. Such methods include, but are not limited to expressionanalysis using nucleic acid chips (e.g. Affymetrix chips) andquantitative RT-PCR based methods using, for example real-time detectionof the transcripts. Analysis of transcript levels according to thepresent invention can be made using total or messenger RNA or proteinsencoded by the genes identified in the diagnostic gene groups of thepresent invention as a starting material. In the preferred embodimentthe analysis is an immunohistochemical analysis with an antibodydirected against proteins comprising at least about 10-20, 20-30,preferably at least 36, at least 36-50, 50, about 50-60, 60-70, 70-80,80-90, 96,100-180, 180-200, 200-250, 250-300, 300-350,350-400, 400-450,450-500, 500-535 proteins encoded by the genes and/or transcripts asshown in Tables 1-7.

The methods of analyzing transcript levels of the gene groups in anindividual include Northern-blot hybridization, ribonuclease protectionassay, and reverse transcriptase polymerase chain reaction (RT-PCR)based methods. The different RT-PCR based techniques are the mostsuitable quantification method for diagnostic purposes of the presentinvention, because they are very sensitive and thus require only a smallsample size which is desirable for a diagnostic test. A number ofquantitative RT-PCR based methods have been described and are useful inmeasuring the amount of transcripts according to the present invention.These methods include RNA quantification using PCR and complementary DNA(cDNA) arrays (Shalon et al., Genome Research 6(7):63945, 1996; Bernardet al., Nucleic Acids Research 24(8): 143542, 1996), real competitivePCR using a MALDI-TOF Mass spectrometry based approach (Ding et al,PNAS, 100: 3059-64, 2003), solid-phase mini-sequencing technique, whichis based upon a primer extension reaction (U.S. Pat. No. 6,013,431,Suomalainen et al. Mol. Biotechnol. June; 15(2): 123-31, 2000), ion-pairhigh-performance liquid chromatography (Doris et al. J. Chromatogr. AMay 8; 806(1):47-60, 1998), and 5′ nuclease assay or real-time RT-PCR(Holland et al. Proc Natl Acad Sci USA 88: 7276-7280, 1991).

Methods using RT-PCR and internal standards differing by length orrestriction endonuclease site from the desired target sequence allowingcomparison of the standard with the target using gel electrophoreticseparation methods followed by densitometric quantification of thetarget have also been developed and can be used to detect the amount ofthe transcripts according to the present invention (see, e.g., U.S. Pat.Nos. 5,876,978; 5,643,765; and 5,639,606.

The samples are preferably obtained from bronchial airways using, forexample, endoscopic cytobrush in connection with a fiber opticbronchoscopy. In one embodiment, the cells are obtained from theindividual's mouth buccal cells, using, for example, a scraping of thebuccal mucosa.

In one preferred embodiment, the invention provides a prognostic and/ordiagnostic immunohistochemical approach, such as a dip-stick analysis,to determine risk of developing lung disease. Antibodies againstproteins, or antigenic epitopes thereof, that are encoded by the groupof genes of the present invention, are either commercially available orcan be produced using methods well know to one skilled in the art.

The invention contemplates either one dipstick capable of detecting allthe diagnostically important gene products or alternatively, a series ofdipsticks capable of detecting the amount proteins of a smallersub-group of diagnostic proteins of the present invention.

Antibodies can be prepared by means well known in the art. The term“antibodies” is meant to include monoclonal antibodies, polyclonalantibodies and antibodies prepared by recombinant nucleic acidtechniques that are selectively reactive with a desired antigen.Antibodies against the proteins encoded by any of the genes in thediagnostic gene groups of the present invention are either known or canbe easily produced using the methods well known in the art. Internetsites such as Biocompare through the World Wide Web at biocompare.com atabmatrix to provide a useful tool to anyone skilled in the art to locateexisting antibodies against any of the proteins provided according tothe present invention.

Antibodies against the diagnostic proteins according to the presentinvention can be used in standard techniques such as Western blotting orimmunohistochemistry to quantify the level of expression of the proteinsof the diagnostic airway proteome. This is quantified according to theexpression of the gene transcript, i.e. the increased expression oftranscript corresponds to increased expression of the gene product, i.e.protein. Similarly decreased expression of the transcript corresponds todecreased expression of the gene product or protein. Detailed guidanceof the increase or decrease of expression of preferred transcripts inlung disease, particularly lung cancer, is set forth in the tables. Forexample, Tables 5 and 6 describe a group of genes the expression ofwhich is altered in lung cancer.

Immunohistochemical applications include assays, wherein increasedpresence of the protein can be assessed, for example, from a saliva orsputum sample.

The immunohistochemical assays according to the present invention can beperformed using methods utilizing solid supports. The solid support canbe a any phase used in performing immunoassays, including dipsticks,membranes, absorptive pads, beads, microtiter wells, test tubes, and thelike. Preferred are test devices which may be conveniently used by thetesting personnel or the patient for self-testing, having minimal or noprevious training. Such preferred test devices include dipsticks,membrane assay systems as described in U.S. Pat. No. 4,632,901. Thepreparation and use of such conventional test systems is well describedin the patent, medical, and scientific literature. If a stick is used,the anti-protein antibody is bound to one end of the stick such that theend with the antibody can be dipped into the solutions as describedbelow for the detection of the protein. Alternatively, the samples canbe applied onto the antibody-coated dipstick or membrane by pipette ordropper or the like.

The antibody against proteins encoded by the diagnostic airwaytranscriptome (the “protein”) can be of any isotype, such as IgA, IgG orIgM, Fab fragments, or the like. The antibody may be a monoclonal orpolyclonal and produced by methods as generally described, for example,in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, 1988, incorporated herein by reference. The antibody can beapplied to the solid support by direct or indirect means. Indirectbonding allows maximum exposure of the protein binding sites to theassay solutions since the sites are not themselves used for binding tothe support. Preferably, polyclonal antibodies are used since polyclonalantibodies can recognize different epitopes of the protein therebyenhancing the sensitivity of the assay.

The solid support is preferably non-specifically blocked after bindingthe protein antibodies to the solid support. Non-specific blocking ofsurrounding areas can be with whole or derivatized bovine serum albumin,or albumin from other animals, whole animal serum, casein, non-fat milk,and the like.

The sample is applied onto the solid support with bound protein-specificantibody such that the protein will be bound to the solid supportthrough said antibodies. Excess and unbound components of the sample areremoved and the solid support is preferably washed so theantibody-antigen complexes are retained on the solid support. The solidsupport may be washed with a washing solution which may contain adetergent such as Tween-20, Tween-80 or sodium dodecyl sulfate.

After the protein has been allowed to bind to the solid support, asecond antibody which reacts with protein is applied. The secondantibody may be labeled, preferably with a visible label. The labels maybe soluble or particulate and may include dyed immunoglobulin bindingsubstances, simple dyes or dye polymers, dyed latex beads,dye-containing liposomes, dyed cells or organisms, or metallic, organic,inorganic, or dye solids. The labels may be bound to the proteinantibodies by a variety of means that are well known in the art. In someembodiments of the present invention, the labels may be enzymes that canbe coupled to a signal producing system. Examples of visible labelsinclude alkaline phosphatase, beta-galactosidase, horseradishperoxidase, and biotin. Many enzyme-chromogen orenzyme-substrate-chromogen combinations are known and used forenzyme-linked assays. Dye labels also encompass radioactive labels andfluorescent dyes.

Simultaneously with the sample, corresponding steps may be carried outwith a known amount or amounts of the protein and such a step can be thestandard for the assay. A sample from a healthy individual exposed to asimilar air pollutant such as cigarette smoke, can be used to create astandard for any and all of the diagnostic gene group encoded proteins.

The solid support is washed again to remove unbound labeled antibody andthe labeled antibody is visualized and quantified. The accumulation oflabel will generally be assessed visually. This visual detection mayallow for detection of different colors, for example, red color, yellowcolor, brown color, or green color, depending on label used. Accumulatedlabel may also be detected by optical detection devices such asreflectance analyzers, video image analyzers and the like. The visibleintensity of accumulated label could correlate with the concentration ofprotein in the sample. The correlation between the visible intensity ofaccumulated label and the amount of the protein may be made bycomparison of the visible intensity to a set of reference standards.Preferably, the standards have been assayed in the same way as theunknown sample, and more preferably alongside the sample, either on thesame or on a different solid support.

The concentration of standards to be used can range from about 1 mg ofprotein per liter of solution, up to about 50 mg of protein per liter ofsolution. Preferably, two or more different concentrations of an airwaygene group encoded proteins are used so that quantification of theunknown by comparison of intensity of color is more accurate.

For example, the present invention provides a method for detecting riskof developing lung cancer in a subject exposed to cigarette smokecomprising measuring the transcription profile in a nasal epithelialcell sample of the proteins encoded by one or more groups of genes ofthe invention in a biological sample of the subject. Preferably at leastabout 30, still more preferably at least about 36, 40, 50, 60, 70, 80,90, 100, 110, 120, 130, 140, 150, 160, 170, or about 180 of the proteinsencoded by the airway transcriptome in a biological sample of thesubject are analyzed. The method comprises binding an antibody againsteach protein encoded by the gene in the gene group (the “protein”) to asolid support chosen from the group consisting of dip-stick andmembrane; incubating the solid support in the presence of the sample tobe analyzed under conditions where antibody-antigen complexes form;incubating the support with an anti-protein antibody conjugated to adetectable moiety which produces a signal; visually detecting saidsignal, wherein said signal is proportional to the amount of protein insaid sample; and comparing the signal in said sample to a standard,wherein a difference in the amount of the protein in the sample comparedto said standard of the same group of proteins, is indicative ofdiagnosis of or an increased risk of developing lung cancer. Thestandard levels are measured to indicate expression levels in an airwayexposed to cigarette smoke where no cancer has been detected.

The assay reagents, pipettes/dropper, and test tubes may be provided inthe form of a kit. Accordingly, the invention further provides a testkit for visual detection of the proteins encoded by the airway genegroups, wherein detection of a level that differs from a pattern in acontrol individual is considered indicative of an increased risk ofdeveloping lung disease in the subject. The test kit comprises one ormore solutions containing a known concentration of one or more proteinsencoded by the airway transcriptome (the “protein”) to serve as astandard; a solution of a anti-protein antibody bound to an enzyme; achromogen which changes color or shade by the action of the enzyme; asolid support chosen from the group consisting of dip-stick and membranecarrying on the surface thereof an antibody to the protein. Instructionsincluding the up or down regulation of the each of the genes in thegroups as provided by the Tables 1 and 2 are included with the kit.

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984. IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The methods of the present invention can employ solid substrates,including arrays in some preferred embodiments. Methods and techniquesapplicable to polymer (including protein) array synthesis have beendescribed in U.S. Ser. No. 09/536,841, WO 00/58516, U.S. Pat. Nos.5,143,854, 5,242,974, 5,252,743, 5,324,633, 5,384,261, 5,405,783,5,424,186, 5,451,683, 5,482,867, 5,491,074, 5,527,681, 5,550,215,5,571,639, 5,578,832, 5,593,839, 5,599,695, 5,624,711, 5,631,734,5,795,716, 5,831,070, 5,837,832, 5,856,101, 5,858,659, 5,936,324,5,968,740, 5,974,164, 5,981,185, 5,981,956, 6,025,601, 6,033,860,6,040,193, 6,090,555, 6,136,269, 6,269,846 and 6,428,752, in PCTApplications Nos. PCT/US99100730 (International Publication Number WO99/36760) and PCT/US01/04285, which are all incorporated herein byreference in their entirety for all purposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptideand protein arrays.

Nucleic acid arrays that are useful in the present invention include,but are not limited to those that are commercially available fromAffymetrix (Santa Clara, Calif.) under the brand name GeneChip7. Examplearrays are shown on the website at affymetrix.com.

Examples of gene expression monitoring, and profiling methods that areuseful in the methods of the present invention are shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Other examples of uses are embodied in U.S. Pat. Nos.5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506:

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with expressionanalysis, the nucleic acid sample may be amplified by a variety ofmechanisms, some of which may employ PCR. See, e.g., PCR Technology:Principles and Applications for DNA Amplification (Ed. H. A. Erlich,Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods andApplications (Eds. Innis, et al., Academic Press, San Diego, Calif.,1990); Mattila et al., Nucleic Acids Res. 19, 4967 (1991); Eckert etal., PCR Methods and Applications 1, 17 (1991); PCR (Eds. McPherson etal., IRL Press, Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195,4,800,159 4,965,188, and 5,333,675, and each of which is incorporatedherein by reference in their entireties for all purposes. The sample maybe amplified on the array. See, for example, U.S. Pat. No. 6,300,070 andU.S. patent application Ser. No. 09/513,300, which are incorporatedherein by reference.

Other suitable amplification methods include the ligase chain reaction(LCR) (e.g., Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NABSA). (U.S. Pat. Nos.5,409,818, 5,554,517, and 6,063,603). Other amplification methods thatmay be used are described in, U.S. Pat. Nos. 5,242,794, 5,494,810,4,988,617 and in U.S. Ser. No. 09/854,317, each of which is incorporatedherein by reference.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described, for example, in Dong etalt, Genome Research 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947,6,391,592 and U.S. patent application Ser. Nos. 09/916,135, 09/920,491,09/910,292, and 10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y, 1989); Berger and Kimmel Methods in Enzymology, Vol.152, Guide to Molecular Cloning Techniques (Academic Press, Inc., SanDiego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described, for example, in U.S. Pat.No. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623 each ofwhich are incorporated herein by reference

The present invention also contemplates signal detection ofhybridization between the sample and the probe in certain embodiments.See, for example, U.S. Pat. Nos. 5,143,854, 5,578,832; 5,631,734;5,834,758; 5,936,324; 5,981,956; 6,025,601; 6,141,096; 6,185,030;6,201,639; 6,218,803; and 6,225,625, in provisional U.S. Patentapplication 60/364,731 and in PCT Application PCT/US99/06097 (publishedas WO99/47964).

Examples of methods and apparatus for signal detection and processing ofintensity data are disclosed in, for example, U.S. Pat. Nos. 5,143,854,5,547,839, 5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092,5,902,723, 5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096,6,185,030, 6,201,639; 6,218,803; and 6,225,625, in U.S. Patentapplication 60/364,731 and in PCT Application PCT/US99/06097 (publishedas WO99/47964).

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Arsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001).

The present invention also makes use of various computer programproducts and software for a variety of purposes, such as, probe design,management of data, analysis, and instrument operation. See, forexample, U.S. Pat. Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164,6,066,454, 6,090,555, 6,185,561, 6,188,783, 6,223,127, 6,229,911 and6,308,170.

Additionally, the present invention may have embodiments that includemethods for providing gene expression profile information over networkssuch as the Internet as shown in, for example, U.S. patent applicationSer. Nos. 10/063,559, 60/349,546, 60/376,003, 60/394,574, 60/403,381.

Throughout this specification, various aspects of this invention arepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible sub-ranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 10-20 should be considered to have specificallydisclosed sub-ranges such as from 10-13, from 10-14, from 10-15, from11-14, from 11-16, etc., as well as individual numbers within thatrange, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. Thisapplies regardless of the breadth of the range. In addition, thefractional ranges are also included in the exemplified amounts that aredescribed. Therefore, for example, a range of 1-3 includes fractionssuch as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, etc. This applies particularly tothe amount of increase or decrease of expression of any particular geneor transcript.

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a reference, for example a patent applicationis cited in the specification, it should be understood that it isincorporated by reference in its entirety for all purposes as well asfor the proposition that is recited.

EXAMPLES Example 1

In this study, we obtained nucleic acid samples (RNA/DNA) from noseepithelial cells. We also obtained nucleic acids from blood to provideone control. We used our findings in the PCT/US2006/014132 to comparethe gene expression profile in the bronchial epithelial cells asdisclosed in the PCT/US2006/014132 to the gene expression patterndiscovered in this example from the nasal epithelial cells.

We have explored the concept that inhaled toxic substances create aepithelial cell “field of injury” that extends throughout therespiratory tract. We have developed the hypothesis that this “field ofinjury”, measured most recently in our laboratory with high density geneexpression arrays, provides information about the degree of airwayexposure to a toxin and the way in which an individual has responded tothat toxin. Our studies have been focused on cigarette smoke, the majorcause of lung cancer and of COPD, although it is likely that mostinhaled toxins result in a change in gene expression of airwayepithelial cells.

We began our studies by examining allelic loss in bronchial epithelialcells brushed from airways during diagnostic bronchcoscopy. We showed,as have others, that allelic loss occurs throughout the intra-pulmonaryairways in smokers with lung cancer, on the side of the cancer as wellas the opposite side from the cancer. Allelic loss also occurs, but to alesser extent, in airway epithelial cells of smokers without cancer(Clinical Cancer Research 5:2025, 1999). We expended these studies toadenocarcinomas from smokers and non-smokers and showed that there was a“field of injury” in non-cancerous lung tissue of smokers, but not innon-smokers (Lung Cancer. 39:23, 2003, Am. J. Respir. Cell. Mol. Biol.29:157, 2003).

We have progressed to using high density arrays to explore patterns ofgene expression that occur in large airway epithelial cells of smokersand non-smokers. We have defined the types of genes that are induced bycigarette smoke, the relation to the amount smoked, racial differences(ATS) in how individuals respond to cigarette smoke, the changes thatare reversible and not reversible in individuals who stop smoking (PNAS.101: 10143-10148, 2004). In addition, we have recently documentedchanges that occur in smokers who develop lung cancer (submitted andAACR), and changes that occur in smokers who develop COPD (Am. J.Respir. Cell Mol. Biol. 31: 601, 2004). All of these studies are ongoingin our laboratory and all depend on obtaining large airway epithelialcells at bronchoscopy, a process that does not lend itself to surveyinglarge populations in epidemiologic studies.

In order to develop a tool that could assay airway epithelial geneexpression without bronchoscopy in large numbers of smokers, we begun toexplore the potential of using epithelial cells obtained from the oralmucosa. We developed a method of obtaining RNA from mouth epithelialcells and could measure expression levels of a few genes that changed inthe bronchial epithelium of smokers, but problems with the quality andquantity of RNA obtained from the mouth has limited widespreadapplication of this method (Biotechniques 36:484-87, 2004).

We have now shown that epithelial cells obtained by brushing the nasalmucosa could be used as a diagnostic and prognostic tool for lungdisorders. Preliminary results show that we can obtain abundant amountsof high quality RNA and DNA from the nose with ease (see protocolbelow), that we can measure gene expression using this RNA and highdensity microarrays and that many of the genes that change with smokingin the bronchial epithelium also change in the nose (see FIG. 1). Wehave further shown that gene expression in nasal epithelium can be usedto define a potentially diagnostic and clinical stage-specific patternof gene expression in subjects with sarcoidosis, even when thesarcoidosis does not clinically involve the lung (see FIG. 2). We canalso obtain DNA from these same specimens allowing us to assess genemethylation patterns and genetic polymorphisms that explain changes ingene expression.

These studies show that gene expression in nasal epithelial cells,obtained in a non-invasive fashion, can indicate individual responses toa variety of inhaled toxins such as cigarette smoke, and can providediagnostic, and possibly prognostic and pathogenetic information about avariety of diseases that involve the lung.

Accordingly, based on our studies we have now developed the method ofanalyzing nasal epithelial cells as a technique and as a screening toolthat can be used to evaluate individual and population responses to avariety of environmental toxins and as a diagnostic/prognostic tool fora variety of lung diseases, including lung cancer. While our initialstudies utilize “discovery-based” genome-wide expression profiling, itis likely that initial studies will ultimately lead to a simpler“defined-gene” platform that will be less complicated and costly andmight be used in the field.

Protocol for Noninvasive Nasal Epithelium RNA and DNA Isolation:

Following local anesthesia with 2% lidocaine solution, a Cytosoft brushis inserted into the right nare and under the inferior turbinate using anasal speculum for visualization. The brush is turned 3 times to collectepithelial cells and immediately placed into RNA Later. Repeat brushingis performed and the 2nd brush is placed in PBS for DNA isolation.

Extending the Airway ‘Field of Injury’ to the Mouth and Nose

While we have demonstrated gene expression differences in bronchialepithelium associated with current, cumulative and past tobaccoexposure, the relatively invasive nature of bronchoscopy makes thecollection of these tissue samples challenging for large scalepopulation studies and for studies of low-disease-risk individuals.Given our hypothesis that the field of tobacco injury extends toepithelial cells lining the entire respiratory tract, we performed apilot study to explore the relationship between bronchial, mouth andnasal gene expression in response to tobacco exposure as nasal and oralbuccal epithelium are exposed to cigarette smoke and can be obtainedusing noninvasive methods. In our pilot study, we collected 15 nasalepithelial samples (8 never smokers, 7 current smokers) via brushing theright inferior turbinate as described in our Research Methods and Designsection. In addition, we collected buccal mucosa epithelial samples from10 subjects (5 never smokers, 5 current smokers) using a scraping devicethat we have described previously [38] (see Appendix). All samples wererun on Affymetrix HG-U133A arrays. Due to the small amounts (1-2 ug) ofpartially degraded RNA obtained from the mouth, samples were collectedserially on each subject monthly and pooled to yield sufficient RNA (6-8ug), Low transcript detection rates were observed for mouth samples,likely as a result of lower levels of intact full-length mRNA in themouth samples

A relationship between the tobacco-smoke induced pattern of geneexpression in all three tissues was first identified by Gene SetEnrichment Analysis (GSEA; [39]) which demonstrates that genesdifferentially expressed in the bronchus are similarly changed in boththe mouth and nose (GSEA p<0.01). We next performed a 2 way ANOVA toidentify 365 genes are differentially expressed with smoking across allthree tissues at p<0.001. PCA of all samples normalized within eachtissue for these 365 genes is shown in FIG. 5.

Finally, while this pilot study in the nose and mouth was not wellpowered for class prediction, we explored the possibility of using thesetissues to identify biomarkers for smoke exposure. The genes with the 20highest and 20 lowest signal-to-noise ratios between smokers andnever-smokers were identified in both the nose and mouth. A classifierwas then trained using these genes in bronchial epithelial samples (15current and 15 never smokers), and tested on an independent test set of41 samples. Genes selected from mouth and nose classify bronchialepithelium of current vs. never-smokers with high accuracy:

Genes Random selected Genes selected Genes selected sselected from Nosefrom Mouth from Bronch Genes Bronchus 82.8% 79.2% 93.2% 64.2 ± 8.1Classification Accuracy

This pilot study established the feasibility of obtaining significantquantities of good quality RNA from brushings of the nasal mucosasuitable for DNA microarray studies and has demonstrated a relationshipbetween previously defined smoking-related changes in the bronchialairway and those occurring in the nasal epithelium. While the qualityand quantity of RNA obtained from buccal mucosa complicates analysis onthe U133A platform, pooled studies suggest a gene-expressionrelationship to the bronchial airway in the setting of tobacco exposure.These results support the central hypothesis that gene expressionprofiles in the upper airway reflect host response to exposure. By usinga novel array platform with the potential to measure gene expression insetting of partially degraded RNA, we propose to more fully explore theability to create biomarkers of tobacco exposure with samples from noseand mouth epithelium.

Example 2 A Comparison of the Genomic Response to Smoking in Buccal,Nasal and Airway Epithelium

Approximately 1.3 billion people smoke cigarettes worldwide whichaccounts for almost 5 million preventable deaths per year (1). Smokingis a significant risk factor for lung cancer, the leading cause ofcancer-related death in the United States, and chronic obstructivepulmonary disease (COPD), the fourth leading cause of death overall.Approximately 90% of lung cancer can be attributed to cigarette smoking,yet only 10-15% of smokers actually develop this disease (2). Despitethe well-established causal role of cigarette smoke in lung cancer andCOPD, the molecular epidemiology explaining why only a minority ofsmokers develop them is still poorly understood.

Cigarette smoking has been found to induce a number of changes in boththe upper and lower respiratory tract epithelia including cellularatypia (3, 4), aberrant gene expression, loss of heterozygosity (3, 5)and promoter hypermethylation. Several authors have reported molecularand genetic changes such as LOH or microsatellite alterations dispersedthroughout the airway epithelium of smokers including areas that arehistologically normal (4, 6). We previously have characterized theeffect of smoking on the normal human airway epithelial transcriptomeand found that smoking induces expression of airway genes involved inregulation of oxidant stress, xenobiotic metabolism, and oncogenesiswhile suppressing those involved in regulation of inflammation and tumorsuppression (7). While this bronchoscopy-based study elucidated somepotential candidates for biomarkers of smoking related lung damage,there is currently a significant impetus to develop less invasiveclinical specimens to serve as surrogates for smoking related lungdamage.

Oral and nasal mucosa are attractive candidates for a biomarkers sincethey are exposed to high concentrations of inhaled carcinogens and aredefinitively linked to smoking-related diseases (8). We have previouslyshown that it is feasible to obtain sufficient RNA from both nasal (9)and buccal mucosa for gene expression analysis (10) despite the highlevel of RNAses in saliva and nasal secretions (11,12). Few studies havecharacterized global gene expression in either of these tissues, andnone has attempted to establish a link between upper and lower airwaygene expression changes that occur with smoking. A pilot study by Smithet. al. used brush biopsies of buccal mucosa from smokers and nonsmokersto obtain RNA for cDNA microarrays and found approximately 100 genesthat could distinguish the two groups in training and test sets. Whilethe study provided encouraging evidence that buccal gene expressionchanges with smoking, many of these genes were undefined ESTs, and thestudy did not address any potential relationship between geneticresponses in the upper and lower airways. Spivak et. al. found aqualitative relationship via PCR (i.e. detected or not detected) betweenpatient matched buccal mucosa and laser-dissected lung epithelial cellsacross nine carcinogen or oxidant-metabolizing genes (13) in 11 subjectsbeing evaluated for lung cancer. However, quantitative real-time PCR ofthese genes in buccal mucosa was not able to reliably predict lungcancer vs. control cases. While global gene expression profiling onnasal brushing has been done recently on children with asthma (14) andcystic fibrosis (15), we are unaware of any studies addressing theeffects of smoking on nasal epithelial gene expression.

In the current study, we report for the first time, a genome wideexpression assay of buccal and nasal mucosa on normal healthyindividuals, which herein are referred to as the “normal buccal andnasal transcriptomes”. We then evaluate the effects of smoking on thesetranscriptomes and compare them to a previous bronchial epithelial geneexpression dataset. By comparing these smoking-induced changes in themouth, nose, and bronchus we establish a relationship between the lowerand upper airway genetic responses to cigarette smoke and furtheradvance the concept of a smoking-induced “field defect” on a global geneexpression level. Lastly, we validate the use of mass spectrometry as afeasible method for multiplexed gene expression studies using smallamounts of degraded RNA from buccal mucosa scrapings.

Study Population

Microarrays were performed on total of 25 subjects and mass spectrometryvalidation on 14 additional subjects. Demographic data for themicroarray and mass spectrometry validation groups are presented inTable 11.

Microarray analysis of normal tissue samples was performed on previouslypublished datasets collected from the Gene Expression Omnibus (GEO).Ninety two samples spanning 10 different tissues types were analyzedaltogether, including 12 nasal and buccal epithelial samples ofnon-smokers collected for this study. Additional microarray data fromnormal nasal epithelial samples were also collected to determine thereproducibility of gene expression patterns in nasal tissue collectedfrom a different study. A detailed breakdown of the different tissuesanalyzed and number of samples within each tissue type are shown inTable 12.

The Relationship Between Normal Airway Epithelial Cells

Principal component analysis (PCA) of the normal tissue samples spanning10 tissue types (n=92 total samples) was performed across the 2382 genescomprising the normal airway transcriptome, which has been previouslycharacterized (Spira et. al, 2004, PNAS). FIG. 7 shows bronchial andnasal epithelial samples clearly grouped together based on theexpression of these 2382 genes.

Overrepresented sets of functional gene categories (“functional sets”)among the 2382 normal airway transcriptome genes were determined by EASEanalysis. Table 13 lists the 16 functional sets that were significantlyoverrepresented among the normal airway transcriptome. On average therewere approximately 109 probe sets per functional cluster. A variabilitymetric was used to determine those functional sets that were mostdifferent across the 10 tissue types. Aldehyde dehydrogenase, antigenprocessing and presentation, and microtubule and cytoskeletal complexwere the most variable functional sets. The least variable sets includedribosomal subunits, and nuclear and protein transport. Two dimensionalhierarchical clustering was also performed on each of these 16functional sets to determine which tissues showed similar expressionpatterns across all the genes in each set. Among the top three mostvariable functional sets listed above, bronchial and nasal epithelialsamples always grouped together (data not shown).

To further examine the relationship between bronchial epithelial tissuesand other tissues, genes from functional groups commonly expressed inairway epithelium were selected from among the normal airwaytranscriptome. Genes from the mucin, dynein, microtubule, keratin,glutathione, cytochrome P450, and aldehyde dehydrogenase functionalgroups were selected from among the 2382 genes in the normal airwaytranscriptome, based on their gene annotations. Fifty-nine genes fromthese functional groups were present among the normal airwaytranscriptome and analyzed using supervised hierarchical clustering, asshown in FIG. 8. Bronchial and nasal epithelial samples clusteredtogether based on the expression of these 59 genes, with many beingexpressed at higher levels in these two tissues. Genes highly expressedin bronchial and nasal epithelium were generally evenly distributedamong the five functional groups. Several dynein, cytochrome P450, andaldehyde dehydrogenase genes were expressed highly in bronchial andnasal epithelium compared to other tissues. Buccal mucosa samplesclustered mainly with lung tissue, with specific keratin genes beinghighly expressed. While some keratins were expressed specifically inskin and esophageal epithelium, other keratins, such as KRT7, KRT8,KRT18, and KRT19 were expressed primarily in bronchial and nasalepithelium. The same pattern was seen with mucin genes, with MUC4,MUC5AC, and MUC16 being expressed primarily in bronchial and nasalepithelium, while MUC1 was expressed in other epithelial tissues.Glutathione genes were expressed highly in bronchial and nasalepithelium as well as other tissues. Microtubule expression was fairlyeven across all tissues.

To explore the similar expression pattern between bronchial and nasalepithelium, a metagene was created by selected a subset of the 59functionally relevant normal transcriptome genes with highly correlatedexpression in between bronchial and nasal samples All genes which werehighly correlated to the metagene (R>0.6, p<0.001) were selected andanalyzed using EASE to determine sets functionally overrepresentedcategories. The microtubule and cytoskeletal complex functional set wassignificantly enriched among the genes most highly correlated with theexpression pattern of the metagene.

A separate set of normal nasal epithelial samples run on the samemicroarray platform (16) was used in place of our nasal epithelialdataset to determine the reproducibility of the relationships in geneexpression between bronchial and nasal epithelium. This separate nasalepithelial dataset consisted of 11 normal epithelial samples run onAffymetrix HG133A microarrays. These samples were first examined withthe 92 normal tissue samples from previous analysis. A correlationmatrix was created to determine the average pearson correlation of eachset of samples within a tissue type with samples from other tissuetypes. The two nasal epithelial datasets had the highest correlationwith each other, with the next highest correlation being between nasaland bronchial epithelial samples. These 11 nasal epithelial samples alsoclustered together with bronchial epithelial samples across the entirenormal transcriptome and the subset of 59 functionally relevant genesfrom the transcriptome when used in place of our original 8 nasalepithelial samples.

Effect of Cigarette Smoking on the Airway Epithelial

To examine the effect of cigarette smoke on airway epithelial cells,current and never smokers samples from buccal and nasal epithelial cellsamples were analyzed together with current and never smokers frombronchial epithelial samples published previously (Spira et. al, 2004,PNAS). In total there were 82 samples across these three tissue types(57 bronch, 10 buccal, 15 nasal). To determine the relationship in theresponse to cigarette smoke between these three tissues, expression of361 genes previously reported to distinguish smokers from non-smokers inbronchial epithelial cells (Spira et. al, 2004, PNAS) was examinedacross all 82 samples from bronchial, nasal, and buccal epithelium.

The 361 genes as shown in Table 8 most differently expressed in theairway epithelial cells of current and never smokers were generally ableto distinguish bronchial, nasal, and buccal epithelial samples based onsmoking status using principal component analysis, with few exceptionsamong buccal mucosa samples (FIG. 3). This finding suggests arelationship between gene expression profiles in epithelial cells in thebronchus and upper airway epithelium in response to cigarette smoke. Tofurther establish this connection across airway epithelial cells, geneset enrichment analysis (GSEA) was performed to determine if genes mostdifferentially expressed in bronchial epithelium based on smoking statuswere overrepresented among the genes that change with smoking in bothnasal and buccal epithelium. We showed that smoking-induced airway genesare significantly enriched among the genes most affected by smoking inbuccal mucosa, with 101 genes composing the “leading edge subset”(p<0.001). The leading edge subset consists of the genes that contributemost to the enrichment of airway genes in buccal mucosa samples. FIG. 6similarly shows that the genes differing most across the bronchialepithelium of smokers were also significantly enriched among the genesmost affected by smoking in nasal epithelial cell samples, with 107genes comprising the leading edge subset (p<0.001). PCA of the leadingedge genes show that they are able to separate buccal mucosa samples andnasal epithelial samples (FIG. 7) based on smoking status, suggesting aglobal relationship in gene expression across airway epithelial cells inresponse to smoking. EASE analysis of the leading edge subsets from FIG.5 reveals that overrepresented functional categories from these genelists include oxidoreductase activity, metal-ion binding, and electrontransport activity (see Table 13).

Study Population

We recruited current and never smoker volunteers from Boston MedicalCenter for a buccal microarray study (n=11), nasal microarray study(n=15) and subsequent prospective buccal epithelial cell massspectrometry validation (n=14). Current smokers in each group had smokedat least 10 cigarettes per day in the past month, with at least acumulative 10 pack-year history. Non-smoking volunteers with significantenvironmental cigarette exposure and subjects with respiratory symptoms,known respiratory, nasal or oral diseases or regular use of inhaledmedications were excluded. For each subject, a detailed smoking historywas obtained including number of pack-years, number of packs per day,age started, and environmental tobacco exposure. Current and neversmokers were matched for age, race and sex. The study was approved bythe Institutional Review Board of Boston Medical Center and all subjectsprovided written informed consent.

Buccal Epithelial Cell Collection

Buccal epithelial cells were collected on 25 subjects (11 for the buccalmicroarray study, 14 for the mass spectrometry validation) as previouslyreported (Spira et. al. 2004, Biotechniques). Briefly, we developed anon-invasive method for obtaining small amounts of RNA from the mouthusing a concave plastic tool with serrated edges. Using gentle pressure,the serrated edge was scraped 5 times against the buccal mucosa on theinside left cheek and placed immediately into 1 mL of RNALATER (Qiagen,Valencia, Calif.). The procedure was repeated for the inside right cheekand the cellular material was combined into one tube. After storage atroom temperature for up to 24 hours, total RNA was isolated from thecell pellet using TRIZOL® reagent (Invitrogen, Carlsbad, Calif.)according to the manufacturer's protocol. The integrity of the RNA wasconfirmed on an RNA denaturing gel. Epithelial cell content wasquantified by cytocentrifugation at 700×g (Cytospin, ThermoShandon,Pittsburgh, Pa.) of the cell pellet and staining with a cytokeratinantibody (Signet, Dedham, Mass.). Using this protocol, we were able toobtain an average of 1823 ng+/−1243 ng of total RNA per collection.Buccal epithelial cells were collected serially over 6 weeks in order toobtain a minimum of 8 ug of RNA per subject. For the 14 subjectsincluded in the mass spectrometry validation, a single collection wassufficient.

Nasal Epithelial Cell Collection

Nasal epithelial cells were collected by first anesthesizing the rightnare with Icc of 1% lidocaine. A nasal speculum (Bionix, Toledo Ohio)was use to spread the nare while a standard cytology brush (CytosoftBrush, Medical Packaging Corporation, Camarillo Calif.) was insertedunderneath the inferior nasal turbinate. The brush was rotated in placeonce, removed, and immediately placed in 1 mL RNA Later (Qiagen,Valencia, Calif.). After storage at 4 degrees overnight, RNA wasisolated via Qiagen RNEASY® Mini Kits per manufacturer's protocol. Asabove, the integrity of RNA was confirmed with an RNA denaturing gel andepithelial cell content was quantified by cytocentrifugation.

Bronchial Epithelial Cell Collection

Bronchial epithelial cells were also obtained on a subset of patients inthe mass spectrometry study (N=6 of the 14) from brushings of the rightmainstem during fibertoptic bronchoscopy with three endoscopiccytobrushes (Cellebrity Endoscopic Cytobrush, Boston Scientific,Boston). After removal of the brush, it was immediately placed inTRIZOL® reagent (Invitrogen), and kept at −80° C. until RNA isolationwas performed. RNA was extracted from the brush using the TRIZOL®reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'sprotocol with an average yield of 8-15 ug of RNA per patient. Integrityof RNA was confirmed by running an RNA-denaturing gel and epithelialcell content was quantified by cytocentrifugation and cytokeratinstaining.

Microarray Data Acquisition and Preprocessing

Eight micrograms of total RNA from buccal epithelial cells (N=11) andnasal epithelial cells (N=15) was processed, labelled, and hybridized toAffymetrix HG-U133A GeneChips containing 22,215 probe sets as previouslydescribed (Spira et. al, 2004, PNAS). A single weighted mean expressionlevel for each gene was derived using MICROARRAY SUITE 5.0 (MAS 5.0)software (Affymetrix, Santa Clara, Calif.). The MAS 5.0 software alsogenerated a detection P value [P(detection)] using a one-sided Wilcoxonsign-ranked test, which indicated whether the transcript was reliablydetected. One buccal mucosa microarray sample was excluded from furtheranalysis based on the percentage of genes detected being lower than twostandard deviations from the median percentage detected across allbuccal mucosa microarray samples, leaving 10 samples for furtheranalysis. All 15 nasal epithelial cell microarray samples containedsufficiently high percentages of genes detected based on the samecriteria, and were all included for further analysis. Microarray datafrom 57 bronchial epithelial cell samples was obtained from previouslypublished data (Spira et. al, 2004, PNAS).

Microarray data from 7 additional normal human tissues was obtained fromdatasets in the Gene Expression Omnibus (GEO). The samples were selectedfrom normal, non-diseased tissue, where there were at least 5 samplesper tissue type. All samples were run on either Affymetrix HGU133A orHGU133 Plus 2.0 microarrays. Array data from normal tissue samples fromthe following 7 tissues were used (GEO accession number included): lung(GSE1650), skin (GSE5667), esophagus (GSE1420), kidney (GSE3526), bonemarrow (GSE3526), heart (GSE2240), and brain (GSE5389). A detailedbreakdown of the array data obtained for these tissues can be seen inTable 12.

Microarray data from buccal mucosa, nasal epithelium, and bronchialepithelial cell samples, as well at normal tissue samples from the 8datasets listed above were each normalized using MAS 5.0, where the meanintensity for each array (excluding the top and bottom 2% of genes) wascorrected using a scaling factor to set the average target intensity ofall probes on the chip to 100. For tissue samples run on the HGU133 Plus2.0 arrays, only those probe sets in common with the HGU133A array wereselected and normalized using MATLAB Student Version 7.1 (The Mathworks,Inc.), where the mean intensity of the selected probes (excluding thetop and bottom 2% of genes) was corrected using a scaling factor to setthe average target intensity of the remaining probes to 100.

Microarray Data Analysis

Clinical information, array data, and gene annotations are stored in aninteractive MYSQL database coded in PERL (37). All statistical analysesdescribed below and within the database were performed using the R v.2.2.0 software (38). The gene annotations used for each probe set werefrom the December 2004 NetAffx HG-U133A annotation files. Principalcomponent analysis (PCA) was performed using the Spotfire DecisionSitesoftware package (39) on the following normal non-smoker tissue samplesfrom 10 different tissue types: bronchial (n=23), nasal (n=8), buccalmucosa (n=5), lung (n=14), skin (n=5), esophagus (n=8), kidney (n=8),bone marrow (n=5), heart (n=5), and brain (n=11). PCA analysis was usedto determine relationships in the gene expression of these tissue typesacross the normal airway transcriptome, which has been previouslycharacterized (Spira et. al, 2004, PNAS).

Functional annotation clustering was performed using the EASE softwarepackage (40) to determine overrepresented sets of functional groups(“functional sets”) among the normal airway transcriptome. Eachfunctional group within a cluster was given a p-value, determined by aFisher-Exact test. The significance of the functional cluster was thendetermined by taking the geometric mean of the p-values of eachfunctional group in the cluster. To limit the number of functional setsreturned by EASE, only functional groups from the Gene Ontology (GO)database below the 5th hierarchical node were used.

To determine the variability of the functional sets across the 10different tissue types, the following formula was used:

V=X ⁻(1 . . . i)[COV(X ⁻ G1 . . . X ⁻ Gk))]

Where Gk is the expression of gene G across all the samples in tissuetype k, i is the total number of genes in a functional cluster, and COVis the coefficient of variation (standard deviation divided by mean) ofthe average expression of gene G across all tissue types. This producedone variability metric (V) for each functional cluster. All the genes ineach functional cluster were then analyzed using 2D hierarchicalclustering performed by using log-transformed z-score normalized datawith a Pearson correlation (uncentered) similarity metric and averagelinkage clustering with CLUSTER and TREEVIEW software (41).

To further analyze the relationship between airway epithelium and othertissue types, genes from the normal airway transcriptome included infunctional categories commonly expressed in airway epithelial cells wereexamined. The functional categories explored were mucin, dynein,microtubule, cytochrome p450, glutathione, aldehyde dehydrogenase, andkeratin. Genes from these categories were determined by selecting allthose genes from the normal airway transcriptome that were also includedin any of these functional groups based on their gene annotation.Fifty-nine genes from the normal airway transcriptome which also spannedthe functional categories of interest were further analyzed across the10 tissues types using supervised hierarchical clustering.

To assess whether genes outside of the normal airway transcriptome wereexpressed at similar levels in bronchial and nasal epithelium, wecreated a metagene by taking a subset of the 59 genes from the normalairway transcriptome spanning the specified functional categories whichwere highly expressed in bronchial and nasal epithelial samples, basedon the Pearson correlation similarity metric for these genes. Acorrelation matrix was then generated between the average expression ofthe metagene across all 10 tissues and each probe set on the HGU133Aarray (22215 total probe sets) across all 10 tissues, to determine geneswith a similar expression pattern to bronchial and nasal epithelium (adetailed protocol for this analysis can be found in the supplement).

A second nasal epithelial dataset (Wright et. al, 2006, Am J Respir CellMol Biol.) was included for further analysis to determine thereproducibility of the expression patterns observed in nasal epitheliumcompared to other tissues. In all there were 11 nasal epithelial samplesfrom this second dataset (GSE2395) which were used in place of ouroriginal 8 nasal samples to determine the reproducibility of geneexpression patterns and relationships between nasal epithelium and othertissues.

To determine the relationship in the response to cigarette smoke bybronchial, buccal, and nasal epithelial cells, PCA was performed across82 smoker and non-smoker samples (57 bronchial, 10 buccal, 15 nasal)using 361 genes differentially expressed between smokers and non-smokersin bronchial epithelial cells (p<0.001), as determined from a priorstudy (Spira et. al, 2004, PNAS). Gene set enrichment analysis (GSEA)(42) was then used to further establish a global relationship betweengene expression profiles from these three tissue types in response tocigarette smoke. Our goal was to determine if the genes mostdifferentially expressed with smoking in bronchial epithelial cells weresignificantly enriched among the top smoking-induced buccal and nasalepithelial genes based on signal-to-noise ratios. P-values weregenerated in GSEA by permuting ranked gene labels and generatingempirical p-values to determine significant enrichment. The airway genesmost significantly enriched among ranked lists of nasal epithelial andbuccal mucosa samples (leading edge subsets), were further analyzedusing PCA to determine the ability of the leading edge subsets todistinguish samples in the nasal and buccal epithelial datasets based onsmoking status.

Table 11 below shows Patient demographic data. Demographic data forpatient samples used for microarray analysis (n=10) and massspectrometry analysis (n=14). *P-values calculated by Fisher Extracttest.

Buccal Microarray Nasal Microarray (N = 10) (N = 15) MS Validation (N =14) Smokers Never P-Value Smokers Never P-Value Smokers Never P-ValueSex 1M, 4F 2M, 3F (p = 0.42*) 6 M, 1 F 5 M, 2 F, (p = .58) 6 M, 1 F 4 M,3 F (p = .24*) 1 U Age 36 (+/− 8) 31 (+/− 9) (p = 0.36) 47 +/− 12 43 +/−18 59 (+/− 15) 41 (+/− 17) (p = 0.06) Race 3 CAU, 2 (p = 0.40*) 3 CAU, 5CAU, 2 5 CAU, 2 4 CAU, (p = .37*) 2 AFA CAU, 3 AFA, 1 AFA, 1 AFA 3AFA 3AFA HIS HIS *P-values calculated by Fisher Extact test

Table 12 below shows breakdown of all microarry datasets analyzed inthis study.

Category Tissue # Samples Platform GEO reference Sample Descriptionepithelial Mouth 5 U133A n/a 5 never smokers epithelial Bronch 23 U133AGSE994 23 never smokers epithelial Nose 8 U133A n/a 8 never smokersepithelial Nose 11 U133A GSE2395 normal nasal epithelium, from cysticfibrosis study epithelial Lung 14 U133A GSE1650 from COPD study, no/mildemphezyma patients epithelial Skin 5 U133A GSE5667 normal skin tissueEpithelial Esophagus 8 U133A GSE1420 normal esophageal epithelium mostlyKidney 8 U133 + 2.0 GSE3526 4 kidney cortex, 4 kidney epithelial medulla(post-mortem) non epithelial Bone 5 U133 + 2.0 GSE3526 5 bone marrow(post- marrow mortem) non epithelial Heart 5 U133A GSE2240 leftventricular myocardium, non-failing non epithelial Brain 11 U133AGSE5389 postmortem orbitofrontal cortex

Table 13 below shows Significantly overrepresented “functional sets”among the normal airway transcriptome. Sixteen functional setssignificantly overrepresented among the normal airway transcriptome,ranked by the variability of each cluster across 10 issue types.

Functional Category Average COV P-value Aldehyde Dehydrogenase108.7083218 0.052807847 Antigen processing and presentation 83.835367680.003259035 Microtubule and Cytoskeletal complex 74.77767675 0.018526945Carbohydrate and Alcohol 67.69528886 0.025158044 catabolism/metabolismOxidative phosphorylation, protein/ion 66.99814067 4.53E−07 transport,metabolism ATPase Activity 62.97844577 7.96E−08 Apoptosis 61.752721950.005467272 Mitochondrial components and activity 61.34998026 3.65E−09NADH Dehydrogenase 58.28368171 4.77E−11 Regulation of protein synthesisand 55.93424773 0.002257705 metabolism NF-kB 55.70796256 0.011130609Protein/macromolecule catabolism 55.62842326 6.74E−05 Intracellular andprotein transport 53.51411018 8.10E−09 Protein/MacromoleculeBiosynthesis 52.28818306 1.62E−25 Vesicular Transport 49.65600620.019136042 Nuclear Transport 44.88736037 0.003807797 Ribosomal Subunits42.57469554 5.42E−15

Table 14 shows Common overrepresented functional categories among“leading edge subsets” from GSEA analysis. Common EASE molecularfunctions of leading edge genes from GSEA analysis. P-values werecalculated using EASE software.

Molecular Function P-value (calculated in EASE) Oxidoreductase activityp < 1.36 × 10−6 Electron transporter activity p < 4.67 × 10−5 Metal ionbinding p < .02 Monooxygenase activity p < .02

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1. A method of diagnosing lung cancer in an individual comprising thesteps of: a) measuring a biological sample comprising nasal epithelialtissue from the individual for the expression of at least 20 genetranscripts from Table 6; b) comparing the expression of the at least 20gene transcripts to a control sample of those transcripts fromindividuals without cancer, wherein increased expression of the genetranscripts as indicated by a negative score in the last column of Table6 and/or decreased expression of the gene transcripts as indicated by apositive score in the last column of Table 6 is indicative of theindividual having lung cancer.
 2. The method of claim 1, wherein atleast 40 gene transcripts are measured.
 3. The method of claim 1,wherein at least 60 gene transcripts are measured.
 4. The method ofclaim 1, wherein at least 70 gene transcripts are measured.
 5. Themethod of claim 1, wherein the gene transcript measured is set forth inTable
 5. 6. The method of claim 1, wherein the gene transcript measuredis set forth in Table
 7. 7. The method of claim 1, wherein the genetranscript measured is set forth in Table 1 wherein the measurement ofthe gene transcript relative to the control uses the third column ofTable 1 setting forth direction of expression in lung cancer todetermine if the individual has lung cancer.
 8. The method of claim 7,wherein the transcript measured is at least Table
 3. 9. The method ofclaim 7, wherein the transcript used is at least the transcripts setforth in Table
 4. 10. A method of diagnosing a lung disease in anindividual exposed to air pollutant comprising the steps of a) measuringexpression profile of a gene group in a nasal epithelial sample from atest individual; and b) comparing the expression profile of the testindividual to an expression profile of a first control individualexposed to similar air pollutant who does not have the lung disease andthe expression profile of a second control individual exposed to similarair pollutants who has the lung disease, wherein similarity of theexpression profile of the test individual with the expression profile ofthe first control rather than that of the second control is indicativeof the test individual being not affected with the lung disease andwherein similarity of the expression profile of the test individual withthe expression profile of the second control individual rather than thatof the first control individual is indicative of the test individualbeing affected or at high risk of developing the lung disease.
 11. Themethod of claim 7, wherein the gene group comprises at least 30,sequences of genes selected from the group consisting with GenBankidentification Nos. NM_(—)003335; NM_(—)000918; NM_(—)006430.1;NM_(—)001416.1; NM_(—)004090; NM_(—)006406.1; NM_(—)003001.2;NM_(—)001319; NM_(—)006545.1; NM_(—)021145.1; NM_(—)002437.1;NM_(—)006286; NM_(—)001003698///NM_(—)001003699///NM_(—)002955;NM_(—)001123///NM_(—)006721; NM_(—)024824; NM_(—)004935.1;NM_(—)002853.1; NM_(—)019067.1; NM_(—)024917.1; NM_(—)020979.1;NM_(—)005597.1; NM_(—)007031.1; NM_(—)009590.1; NM_(—)020217.1;NM_(—)025026.1; NM_(—)014709.1; NM_(—)014896.1; AF010144;NM_(—)005374.1; NM_(—)001696; NM_(—)005494///NM_(—)058246;NM_(—)006534///NM_(—)181659; NM_(—)006368; NM_(—)002268///NM_(—)032771;NM_(—)014033; NM_(—)016138; NM_(—)007048///NM_(—)194441; NM_(—)006694;NM_(—)000051///NM_(—)138292///NM_(—)138293;NM_(—)000410///NM_(—)139002///NM_(—)139003///NM_(—)139004///NM_(—)139005///NM_(—)139006///NM_(—)139007///NM_(—)139008///NM_(—)139009///NM_(—)139010///NM_(—)139011;NM_(—)004691; NM_(—)012070///NM_(—)139321///NM_(—)139322; NM_(—)006095;AI632181; AW024467; NM_(—)021814; NM_(—)005547.1; NM_(—)203458;NM_(—)015547///NM_(—)147161; AB007958.1; NM_(—)207488;NM_(—)005809///NM_(—)181737///NM_(—)181738; NM_(—)016248///NM_(—)144490;AK022213.1; NM_(—)005708; NM_(—)207102; AK023895;NM_(—)144606///NM_(—)144997; NM_(—)018530; AK021474; U43604.1; AU147017;AF222691.1; NM_(—)015116;NM_(—)001005375///NM_(—)001005785///NM_(—)001005786///NM_(—)004081///NM_(—)020363///NM_(—)020364///NM_(—)020420;AC004692; NM_(—)001014; NM_(—)000585///NM_(—)172174///NM_(—)172175;NM_(—)054020///NM_(—)172095///NM_(—)172096///NM_(—)172097; BE466926;NM_(—)018011; NM_(—)024077; NM_(—)012394;NM_(—)019011///NM_(—)207111///NM_(—)207116; NM_(—)017646; NM_(—)021800;NM_(—)016049; NM_(—)014395; NM_(—)014336; NM_(—)018097; NM_(—)019014;NM_(—)024804; NM_(—)018260; NM_(—)018118; NM_(—)014128; NM_(—)024084;NM_(—)005294; AF077053; NM_(—)138387; NM_(—)024531; NM_(—)000693;NM_(—)018509; NM_(—)033128; NM_(—)020706; AI523613; and NM_(—)014884.12. The method of claim 1, wherein the gene group comprises sequences ofgenes selected from the group consisting of genes with GenBankidentification Nos. NM_(—)007062.1; NM_(—)001281.1; BC000120.1;NM_(—)014255.1; BC002642.1; NM_(—)000346.1; NM_(—)006545.1; BG034328;NM_(—)021822.1; NM_(—)021069.1; NM_(—)019067.1; NM_(—)017925.1;NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1; AF126181.1; U93240.1;U90552.1; AF151056.1; U85430.1; U51007.1; BC005969.1; NM_(—)002271.1;AL566172; AB014576.1; BF218804; AK022494.1; AA114843; BE467941;NM_(—)003541.1; R83000; AL161952.1; AK023843.1; AK021571.1; AK023783.1;AU147182; AL080112.1; AW971983; AI683552; NM_(—)024006.1; AK026565.1;NM_(—)014182.1; NM_(—)021800.1; NM_(—)016049.1; NM_(—)019023.1;NM_(—)021971.1; NM_(—)014128.1; AK025651.1; AA133341; and AF198444.1.13. The method of claim 1, wherein the gene group comprises sequences ofgenes selected from the group consisting of genes with GenBankidentification Nos. NM_(—)007062.1; NM_(—)001281.1; BC002642.1;NM_(—)000346.1; NM_(—)006545.1; BG034328; NM_(—)019067.1;NM_(—)017925.1; NM_(—)017932.1; NM_(—)030757.1; NM_(—)030972.1;NM_(—)002268///NM_(—)032771; NM_(—)007048///NM_(—)194441; NM_(—)006694;U85430.1; NM_(—)004691; AB014576.1; BF218804; BE467941; R83000;AL161952.1; AK023843.1; AK021571.1; AK023783.1; AL080112.1; AW971983;AI683552; NM_(—)024006.1; AK026565.1; NM_(—)014182.1; NM_(—)021800.1;NM_(—)016049.1; NM_(—)021971.1; NM_(—)014128.1; AA133341; and AF198444.114. The method of claim 1, wherein the group comprises sequences ofgenes selected from the group consisting of genes with GenBank orUnigene identification Nos. NM_(—)030757.1; R83000; AK021571.1;NM_(—)014182.1; NM_(—)17932.1; U85430.1; AI683552; BC002642.1; AW024467;NM_(—)030972.1; BC021135.1; AL161952.1; AK026565.1; AK023783.1;BF218804; NM_(—)001281.1; NM_(—)024006.1; AK023843.1; BC001602.1;BC034707.1; BC064619.1; AY280502.1; BC059387.1; AF135421.1; BC061522.1;L76200.1; U50532.1; BC006547.2; BC008797.2; BC000807.1; AL080112.1;BC033718.1///BC046176.1///BC038443.1; NM_(—)000346.1; BC008710.1;Hs.288575 (UNIGENE ID); AF020591.1; BC000423.2; BC002503.2; BC008710.1;BC009185.2; Hs.528304 (UNIGENE ID); U50532.1; BC013923.2; BC031091;NM_(—)007062; Hs.249591 (Unigene ID); BC075839.1///BC073760.1;BC072436.1///BC004560.2; BC001016.2; Hs.286261 (Unigene ID); AF348514.1;BC005023.1; BC066337.1///BC058736.1///BC050555.1; Hs.216623 (UnigeneID); BC072400.1; BC041073.1; U43965.1; BC021258.2; BC016057.1;BC016713.1///BC014535.1///AF237771.1; BC000360.2; BC007455.2;BC000701.2; BC010067.2; BC023528.2///BC047680.1; BC064957.1; Hs.156701(Unigene ID); BC030619.2; BC008710.1; U43965.1; BC066329.1; Hs.438867(Unigene ID); BC035025.2///BC050330.1; BC023976.2;BC074852.2///BC074851.2; Hs.445885 (Unigene ID);BC008591.2///BC050440.1///; BC048096.1; AF365931.1; AF257099.1; andBC028912.1.
 15. The method of claim 1, wherein the group comprisessequences of genes selected from the group consisting of genes withGenBank or Unigene identification Nos: NM_(—)003335; NM_(—)001319;NM_(—)021145.1; NM_(—)001003698///NM_(—)001003699///; NM_(—)002955;NM_(—)002853.1; NM_(—)019067.1; NM_(—)024917.1; NM_(—)020979.1;NM_(—)005597.1; NM_(—)007031.1; NM_(—)009590.1; NM_(—)020217.1;NM_(—)025026.1; NM_(—)014709.1; NM_(—)014896.1; AF010144;NM_(—)005374.1; NM_(—)006534///NM_(—)181659; NM_(—)014033; NM_(—)016138;NM_(—)007048///NM_(—)194441; NM_(—)000051///NM_(—)138292///NM_(—)138293;NM_(—)000410///NM_(—)139002///NM_(—)139003///NM_(—)139004///NM_(—)139005///NM_(—)139006///NM_(—)139007///NM_(—)139008///NM_(—)139009///NM_(—)139010///NM_(—)139011;NM_(—)012070///NM_(—)139321///NM_(—)139322; NM_(—)006095; AI632181;AW024467; NM_(—)021814; NM_(—)005547.1; NM_(—)203458;NM_(—)015547///NM_(—)147161; AB007958.1; NM_(—)207488;NM_(—)005809///NM_(—)181737///NM_(—)181738; NM_(—)016248///NM_(—)144490;AK022213.1; NM_(—)005708; NM 207102; AK023895;NM_(—)144606///NM_(—)144997; NM_(—)018530; AK021474; U43604.1; AU147017;AF222691.1; NM_(—)015116;NM_(—)001005375///NM_(—)001005785///NM_(—)001005786///NM_(—)004081///NM_(—)020363///NM_(—)020364///NM_(—)020420;AC004692; NM_(—)001014; NM_(—)000585///NM_(—)172174///NM_(—)172175;NM_(—)054020///NM_(—)172095///NM_(—)172096///NM_(—)172097; BE466926;NM_(—)018011; NM_(—)024077; NM_(—)019011///NM 207111///NM_(—)207116;NM_(—)017646; NM_(—)014395; NM_(—)014336; NM_(—)018097; NM_(—)019014;NM_(—)024804; NM_(—)018260; NM_(—)018118; NM_(—)014128; NM_(—)024084;NM_(—)005294; AF077053; NM_(—)000693; NM_(—)033128; NM_(—)020706;AI523613; and NM_(—)014884, and wherein decrease in expression of atleast S of these genes is indicative of the individual being affectedwith a lung disease.
 16. The method of claim 1, wherein the groupcomprises sequences of genes selected from the group consisting of geneswith GenBank or Unigene identification Nos NM_(—)030757.1; R83000;AK021571.1; NM_(—)17932.1; U85430.1; AI683552; BC002642.1; AW024467;NM_(—)030972.1; BC021135.1; AL161952.1; AK026565.1; AK023783.1;BF218804; AK023843.1; BC001602.1; BC034707.1; BC064619.1; AY280502.1;BC059387.1; BC061522.1; U50532.1; BC006547.2; BC008797.2; BC000807.1;AL080112.1; BC033718.1///BC046176.1///; BC038443.1; Hs.288575 (UNIGENEID); AF020591.1; BC002503.2; BC009185.2; Hs.528304 (UNIGENE ID);U50532.1; BC013923.2; BC031091; Hs.249591 (Unigene ID); Hs.286261(Unigene ID); AF348514.1; BC066337.1///BC058736.1///BC050555.1;Hs.216623 (Unigene ID); BC072400.1; BC041073.1; U43965.1; BC021258.2;BC016057.1; BC016713.1///BC014535.1///AF237771.1; BC000701.2;BC010067.2; Hs.156701 (Unigene ID); BC030619.2; U43965.1; Hs.438867(Unigene ID); BC035025.2///BC050330.1; BC074852.2///BC074851.2;Hs.445885 (Unigene ID); AF365931.1; and AF257099.1, and wherein decreasein expression of at least 5 of these genes is indicative of theindividual being affected with a lung disease.
 17. The method of claim1, wherein the group comprises sequences of genes selected from thegroup consisting of genes with GenBank or Unigene identification NosBF218804; AK022494.1; AA114843; BE467941; NM_(—)003541.1; R83000;AL161952.1; AK023843.1; AK021571.1; AK023783.1; AU147182; AL080112.1;AW971983; AI683552; NM_(—)024006.1; AK026565.1; NM_(—)014182.1;NM_(—)021800.1; NM_(—)016049.1; NM_(—)019023.1; NM_(—)021971.1;NM_(—)014128.1; AK025651.1; AA133341; and AF198444.1, and whereindecrease in expression of at least 5 of these genes is indicative of theindividual being affected with a lung disease.
 18. The method of claim1, wherein the group comprises sequences of genes selected from thegroup consisting of genes with GenBank or Unigene identification NosNM_(—)000918; NM_(—)006430.1; NM_(—)001416.1; NM_(—)004090;NM_(—)006406.1; NM_(—)003001.2; NM_(—)006545.1; NM_(—)002437.1;NM_(—)006286; NM_(—)001123///NM_(—)006721; NM_(—)024824; NM_(—)004935.1;NM_(—)001696; NM_(—)005494///NM_(—)058246; NM_(—)006368;NM_(—)002268///NM_(—)032771; NM_(—)006694; NM_(—)004691; NM_(—)012394;NM_(—)021800; NM_(—)016049; NM_(—)138387; NM_(—)024531; andNM_(—)018509, and wherein increase in expression of at least 5 of thesegenes is indicative of the individual being affected with a lungdisease.
 19. The method of claim 1, wherein the group comprisessequences of genes selected from the group consisting of genes withGenBank or Unigene identification Nos NM_(—)014182.1; NM_(—)001281.1;NM_(—)024006.1; AF135421.1; L76200.1; NM_(—)000346.1; BC008710.1;BC000423.2; BC008710.1; NM_(—)007062; BC075839.1///BC073760.1;BC072436.1///BC004560.2; BC001016.2; BC005023.1; BC000360.2; BC007455.2;BC023528.2///BC047680.1; BC064957.1; BC008710.1; BC066329.1; BC023976.2;BC008591.2///BC050440.1///BC048096.1; and BC028912.1, and whereinincrease in expression of at least 5 of these genes is indicative of theindividual being affected with a lung disease.
 20. The method of claims4-7, wherein the group comprises sequences of genes selected from thegroup consisting of genes with GenBank or Unigene identification NosNM_(—)007062.1; NM_(—)001281.1; BC000120.1; NM_(—)014255.1; BC002642.1;NM_(—)000346.1; NM_(—)006545.1; BG034328; NM_(—)021822.1;NM_(—)021069.1; NM_(—)019067.1; NM_(—)017925.1; NM_(—)017932.1;NM_(—)030757.1; NM_(—)030972.1; AF126181.1; U93240.1; U90552.1;AF151056.1; U85430.1; U51007.1; BC005969.1; NM_(—)002271.1; AL566172;and AB014576.1, and wherein increase in expression of at least 5 ofthese genes is indicative of the individual being affected with a lungdisease.
 21. The method of claim 1, wherein the group comprises 5-9sequences of genes selected from group I and group 2, group I consistingof genes with GenBank or Unigene identification Nos. NM_(—)003335;NM_(—)001319; NM_(—)021145.1; NM_(—)001003698///NM_(—)001003699///;NM_(—)002955; NM_(—)002853.1; NM_(—)019067.1; NM_(—)024917.1;NM_(—)020979.1; NM_(—)005597.1; NM_(—)007031.1; NM_(—)009590.1;NM_(—)020217.1; NM_(—)025026.1; NM_(—)014709.1; NM_(—)014896.1;AF010144; NM_(—)005374.1; NM_(—)006534///NM_(—)181659; NM_(—)014033;NM_(—)016138; NM_(—)007048///NM_(—)194441;NM_(—)000051///NM_(—)138292///NM_(—)138293;NM_(—)000410///NM_(—)139002///NM_(—)139003///NM_(—)139004///NM_(—)139005///NM_(—)139006///NM_(—)139007///NM_(—)139008///NM_(—)139009///NM_(—)139010///NM_(—)139011;NM_(—)012070///NM_(—)139321///NM_(—)139322; NM_(—)006095; AI632181;AW024467; NM_(—)021814; NM_(—)005547.1; NM_(—)203458;NM_(—)015547///NM_(—)147161; AB007958.1; NM_(—)207488;NM_(—)005809///NM_(—)181737///NM_(—)181738; NM_(—)016248///NM_(—)144490;AK022213.1; NM_(—)005708; NM 207102; AK023895;NM_(—)144606///NM_(—)144997; NM_(—)018530; AK021474; U43604.1; AU147017;AF222691.1; NM_(—)015116;NM_(—)001005375///NM_(—)001005785///NM_(—)001005786///NM_(—)004081///NM_(—)020363///NM_(—)020364///NM_(—)020420;AC004692; NM_(—)001014; NM_(—)000585///NM_(—)172174///NM_(—)172175;NM_(—)054020///NM_(—)172095///NM_(—)172096///NM_(—)172097; BE466926;NM_(—)018011; NM_(—)024077; NM_(—)019011///NM_(—)207111///NM_(—)207116;NM_(—)017646; NM_(—)014395; NM_(—)014336; NM_(—)018097; NM_(—)019014;NM_(—)024804; NM_(—)018260; NM_(—)018118; NM_(—)014128; NM_(—)024084;NM_(—)005294; AF077053; NM_(—)000693; NM_(—)033128; NM_(—)020706;AI523613; and NM_(—)014884, and group 2 consisting of genes with GenBankor Unigene identification Nos. NM_(—)000918; NM_(—)006430.1;NM_(—)001416.1; NM_(—)004090; NM_(—)006406.1; NM_(—)003001.2;NM_(—)006545.1; NM_(—)002437.1; NM_(—)006286;NM_(—)001123///NM_(—)006721; NM_(—)024824; NM_(—)004935.1; NM_(—)001696;NM_(—)005494///NM_(—)058246; NM_(—)006368; NM_(—)002268///NM_(—)032771;NM_(—)006694; NM_(—)004691; NM_(—)012394; NM_(—)021800; NM_(—)016049;NM_(—)138387; NM_(—)024531; and NM_(—)018509, and a group of at least 20genes selected from group 3 and group 4, group 3 consisting of geneswith GenBank or Unigene identification Nos BF218804; AK022494.1;AA114843; BE467941; NM_(—)003541.1; R83000; AL161952.1; AK023843.1;AK021571.1; AK023783.1; AU147182; AL080112.1; AW971983; AI683552;NM_(—)024006.1; AK026565.1; NM_(—)014182.1; NM_(—)021800.1;NM_(—)016049.1; NM_(—)019023.1; NM_(—)021971.1; NM_(—)014128.1;AK025651.1; AA133341; and AF198444.1 and group 4 consisting of geneswith GenBank or Unigene identification Nos. NM_(—)007062.1;NM_(—)001281.1; BC000120.1; NM_(—)014255.1; BC002642.1; NM_(—)000346.1;NM_(—)006545.1; BG034328; NM_(—)021822.1; NM_(—)021069.1;NM_(—)019067.1; NM_(—)017925.1; NM_(—)017932.1; NM_(—)030757.1;NM_(—)030972.1; AF126181.1; U93240.1; U90552.1; AF151056.1; U85430.1;U51007.1; BC005969.1; NM_(—)002271.1; AL566172; and AB014576.1.
 22. Themethod of claim 20, wherein decrease in the expression of any one of thegroup I genes and increase in the expression of any one of the group 2genes, and decrease of the group 3 genes and increase of the group 4genes is indicative of the individual being affected with a lungdisease.
 23. The method of claim 1, wherein the group comprises 5-9sequences of genes selected from group 5 and group 6, group 5 consistingof genes with GenBank or Unigene identification Nos. NM_(—)030757.1;R83000; AK021571.1; NM_(—)17932.1; U85430.1; AI683552; BC002642.1;AW024467; NM_(—)030972.1; BC021135.1; AL161952.1; AK026565.1;AK023783.1; BF218804; AK023843.1; BC001602.1; BC034707.1; BC064619.1;AY280502.1; BC059387.1; BC061522.1; U50532.1; BC006547.2; BC008797.2;BC000807.1; AL080112.1; BC033718.1///BC046176.1///; BC038443.1;Hs.288575 (UNIGENE ID); AF020591.1; BC002503.2; BC009185.2; Hs.528304(UNIGENE ID); U50532.1; BC013923.2; BC031091; Hs.249591 (Unigene ID);Hs.286261 (Unigene ID); AF348514.1;BC066337.1///BC058736.1///BC050555.1; Hs.216623 (Unigene ID);BC072400.1; BC041073.1; U43965.1; BC021258.2; BC016057.1;BC016713.1///BC014535.1///AF237771.1; BC000701.2; BC010067.2; Hs.156701(Unigene ID); BC030619.2; U43965.1; Hs.438867 (Unigene ID);BC035025.2///BC050330.1; BC074852.2///BC074851.2; Hs.445885 (UnigeneID); AF365931.1; and AF257099.1, and group 6 consisting of genes withGenBank or Unigene identification Nos. NM_(—)014182.1; NM_(—)001281.1;NM_(—)024006.1; AF135421.1; L76200.1; NM_(—)000346.1; BC008710.1;BC000423.2; BC008710.1; NM_(—)007062; BC075839.1///BC073760.1;BC072436.1///BC004560.2; BC001016.2; BC005023.1; BC000360.2; BC007455.2;BC023528.2/BC047680.1; BC064957.1; BC008710.1; BC066329.1; BC023976.2;BC008591.2///BC050440.1///; BC048096.1; and BC028912.1, and a group ofat least 20 genes selected from group 3 and group 4, group 3 consistingof genes with GenBank or Unigene identification Nos BF218804;AK022494.1; AA114843; BE467941; NM_(—)003541.1; R83000; AL161952.1;AK023843.1; AK021571.1; AK023783.1; AU147182; AL080112.1; AW971983;AI683552; NM_(—)024006.1; AK026565.1; NM_(—)014182.1; NM_(—)021800.1;NM_(—)016049.1; NM_(—)019023.1; NM_(—)021971.1; NM_(—)014128.1;AK025651.1; AA133341; and AF198444.1 and group 4 consisting of geneswith GenBank or Unigene identification Nos. NM_(—)007062.1;NM_(—)001281.1; BC000120.1; NM_(—)014255.1; BC002642.1; NM_(—)000346.1;NM_(—)006545.1; BG034328; NM_(—)021822.1; NM_(—)021069.1;NM_(—)019067.1; NM_(—)017925.1; NM_(—)017932.1; NM_(—)030757.1;NM_(—)030972.1; AF126181.1; U93240.1; U90552.1; AF151056.1; U85430.1;U51007.1; BC005969.1; NM_(—)002271.1; AL566172; and AB014576.1, whereindecrease in the expression of the group 5 genes and increase in theexpression of the group 6 genes, and decrease the group 3 genes andincrease of the group 4 genes is indicative of the individual beingaffected with a lung disease.
 24. The method of claim 1, wherein thetranscripts are selected from the group consisting of transcriptsencoding CYP1B1; AKR1B10; CYP1B1; CYP1A1; CYP1B1; CEACAM5; ALDH3A1;SLC7A11; AKR1C2; NQO1; NQO1; GPX2; MUC5AC; AKR1C2; MUC5AC; AKR1C1;CLDN10; AKR1C3; NQO1; SLC7A11; HGD///LOC642252; AKR1C1; PIR; CYP4F11;TCN1; TM4SF1; KRT14; ME1; CBR1; ADH7; SPDEF; ME1; CXCL14; SRPX2; UPK1B;TRIM16///TRIM16L///LOC653524; KLF4; TXN; TKT; DEFB1; CSTA; CEACAM6;TALDO1; CA12; GCLM; PGD; TXNRD1; CEACAM6; GCLC; GPC1; TFF1; CABYR; CA12;UPK1B; GALNT6; TKT; TSPAN8; UGT1A10///UGT1A8///UGT1A7///UGT1A6///UGT1A;SPDEF; MSMB; ANXA3; MUC5AC; CTGF; IDS; CA12; FTH1; HN1; DPYSL3; GMDS;UGT1A10///UGT1A8///UGT1A7///UGT1A6///UGT1A; ABHD2; GCLC; GALNT7; MSMB;HTATIP2; UGT1A10///UGT1A8///UGT1A7///UGT1A6///UGT1A; S100A10;DAZ1///DAZ3///DAZ2///DAZ4; IDS; PRDX1; CYP4F3;UGT1A10///UGT1A8///UGT1A7///UGT1A6///UGT1A; AGR2; S100P; NDUFA7; MAFG;ZNF323; AP2B1; UGT1A6; NKX3-1; SEPX1; CTSC; GCNT3; GULP1; LOC283677;SMPDL3A; SLC35A3; WBP5; TARS; EIF2AK3; C11orf32; GALNT12; VPS13D;BCL2L13; IMPA2; GMDS; AZGP1; PLCE1; FOLH1; NUDT4///NUDT4P1; TAGLN2; GNE;TSPAN13; GALNT3; HMGN4; SCP2; PLA2G10; GULP1; DIAPH2; RAP1GAP; FTH1;LYPLA1; CREB3L1; AKR1B1; RAB2; SCGB2A1; KIAA0367; ABCC1; TPARL; ABHD2;TSPAN1; DHRS3; ABCC1; FKBP11; TTC9; GSTM3; S100A14; SLC35A1; ENTPD4;P4HB; AGTPBP1; NADK; B4GALT5; CCPG1; PTP4A1; DSG2; CCNG2; CPNE3;SEC31L1; SLC3A2; ARPC3; CDC14B; SLC17A5; H1ST1H2AC; CBLB; H1ST1H2BK;TOM1L1; TIMP1; ABCB6; GFPT1; TIAM1; SORL1; PAM; NADK; RND3; XPOT;SERINC5; GSN; HIGD1A; PDIA3; C3orf14; PRDX4; RAB7; GPR153; ARL1; IDS;GHITM; RGC32; TMED2; PTS; GTF3C1; IDH1; LAMP2; ACTL6A; RAB11A; COX5A;APLP2; PTK9; UBE2J1; TACSTD2; PSMD14; PDIA4; MTMR6; FA2H; NUDT4;TBC1D16; PIGP; CCDC28A; AACS; CHP; TJP2; EFHD2; KATNB1; SPA17; TPBG;GALNT1; HSP90B1; TMED10; SOD1; BECN1; C14orf1; COPB2; TXNDC5; SSR4;TLE1; TXNL1; LRRC8D; PSMB5; SQSTM1; ETHE1; RPN2; TIPARP; CAP1; LOC92482;FKBP1A; EDEM1; CANX; TMEM59; GUK1; LOC57228; SPINT2; C20orf111; ECOP;JTB; REXO2; UFDIL; DDX17; SSH3; TRIOBP; GGA1; FAM53C; PPP3CC; SFRS14;ACTN1; SPEN; CYP2J2; TLE2; ProSAPiP1; PFTK1; PCDH7; FLNB; SIX2; CD81;ZNF331; AMACR; GNB5; CUGBP1; EDD1; TLR5; MGLL; CHST4; SERPINI2; PPAP2B;BCL11A; STEAP3; SYNGR1; CRYM; RUTBC1; PARVA; NFIB; TCF7L1; MAGI2;CCDC81; COL9A2; CNKSR1; NCOR2; INHBB; PEX14; TSPAN9; RAB6B; GSTM5;FLJ10159; TNS1; MT2A; TNFSF13///TNFSF12-TNFSF13; I-Mar; ELF5; JAG2;FLJ23191; PHGDH; CYP2F1; TNS3; GAS6; CD302; PTPRM; CCND1;TNFSF13///TNFSF12-TNFSF13; ADCY2; CCND2; MT1X; SNED1; SFRS14; ANXA6;HNMT; AK1; EPOR; EPAS1; PDE8B; CYFIP2; SLIT1; ACCN2; KAL1; MTIE; MTIF;HLF; SITPEC; JAG2; HSPA2; LOC650610; KRT15; SORD; ITM2A; PEC1; HPGD;CKB; HLF; CYP2A6///CYP2A7///CYP2A7P1///CYP2A13; C14orf132; MT1G; FGFR3;PROS1; FAM107A; MT1X; FXYD1; MTIF; CX3CL1; CX3CL1; CYP2A6; HLF; SLIT2;BCAM; FMO2; MT1H; FLRT3; PRG2; TMEM45A; MMP10; C3///LOC653879; CYP2W1;FABP6; SCGB1A1; MUC5B///LOC649768; FAM107A; SEC14L3; 210524_x_at;213169_at; 212126_at; 43511_s_at; 213891_s_at; 212233_at; and 217626_at.25. The method of claim 1, wherein the transcripts are selected from thegroup consisting of AACS; ABHD2; ADCY2; ADH7; ALDH3A1; AP2B1; APLP2;ARHE; ARL1; ARPC3; ASM3A; AZGP1; C14orf1; C1orf8; CANX; CAP1; CCND2;CCNG2; CEACAM5; CEACAM6; CHP; CLDN10; COX5A; CPNE3; CPR8; CTSC; CYPIA1;CYP2F1; CYP4F11; CYP4F3; DAZ4; DCL-1; DKFZP434J214; DPYSL3; ERP70;FKBP11; FKBP1A; FLJ13052; FOLH1; FTH1; GALNT1; GALNT12; GALNT3; GALNT7;GCLM; GCNT3; GFPT1; GMDS; GNE; GRP58; GSN; HGD; H1ST1H2BK; HMGN4;HTATIP2; IDS; IMPA2; JTB; KATNB1; KDELR3; KIAA0227; KIAA0367; KIAA0905;KLF4; LAMP2; LOC92689; LRRC5; ME1; MSMB; MTIG; MUC5B; NKX3-1; NQO1;NUDT4; OASIS; P4HB; PDEF; PIR; PLA2G10; PPP3CC; PRDX4; RAB11A; RAB2;RAP1GA1; RGC32; RNP24; S100A10; SCGB2A1; SDR1; SEPX1; SLC17A5; SLC35A1;SLC7A11; TACSTD2; TAGLN2; TCN1; TIMP1; TKT; TM4SF13; TM4SF3; TMP21;TXNDC5; UBE2J1; UGT1A10; UPK1B; CYP1B1 and 203369_x_at.
 26. The methodof claim 1, wherein the transcripts are selected from the groupconsisting of transcripts encoding for AACS; ABHD2; ADCY2; ADH7;ALDH3A1; AP2B1; APLP2; ARHE; ARL1; ARPC3; ASM3A; AZGP1; C14orf1; C1orf8;CANX; CAP1; CCND2; CCNG2; CEACAM5; CEACAM6; CHP; CLDN10; COX5A; CPNE3;CPR8; CTSC; CYP1A1; CYP2F1; CYP4F11; CYP4F3; DAZ4; DCL-1; DKFZP434J214;DPYSL3; ERP70; FKBP11; FKBP1A; FLJ13052; FOLH1; FTH1; GALNT1; GALNT12;GALNT3; GALNT7; GCLM; GCNT3; GFPT1; GMDS; GNE; GRP58; GSN; HGD;H1ST1H2BK; HMGN4; HTATIP2; IDS; IMPA2; JTB; KATNB1; KDELR3; KL1A0227;KIAA0367; KIAA0905; KLF4; LAMP2; LOC92689; LRRC5; ME1; MSMB; MTIG;MUCSB; NKX3-1; NQO I; NUDT4; OASIS; P4HB; PDEF; PIR; PLA2G10; PPP3CC;PRDX4; RAB11A; RAB2; RAPIGA1; RGC32; RNP24; S100A10; SCGB2A1; SDR1;SEPX1; SLC17A5; SLC35A1; SLC7A11; TACSTD2; TAGLN2; TCN1; TIMP1; TKT;TM4SF13; TM4SF3; TMP21; TXNDC5; UBE2J1; UGT1A10; UPK1B; CYP1B1 and203369_x_at.
 27. The method of claim 1, wherein the transcripts areselected from the group consisting of transcripts encoding for MUC1,CD164; MUC16; MUC4; MUC5AC; CYP2A6; CYP2B7P1; CYP4B1; POR; CYP2F1;DNAI2; DYNLT1; DNALI1; DNAI1; DNAH9; DNAH7; DYNC112; DYNC1H1; DYNLL1;DYNLRB1; ESD; GSTM2; GSTM1; GSTK1; GSTA1; GPX4; GPX1; MGST2; GSTP1; GSS;GSTO1; KRTI9; KRT7; KRT8; KRT18; KRT10; KRT10; KRT17; KRT5; KRT15;MAPIA; MAPRE1; EML2; MAST4; MACF1; ALDH3A1; ALDH1A1; ALDH3B1; ALDH3B1;ALDH3A2; ALDH1L1; ALDH9A1; ALDH2; K-ALPHA-1; TUBB3; TUBGCP2; TBCA;TUBB2A; TUBA4; TUBB2C; TUBA3; TUBA6; K-ALPHA-1; TUBB; TUBA6; TUBA1;TUBB; K-ALPHA-1; 76P; TUBB3; and TUBB2C.